Anti-interleukin-23 p19 antibodies and methods of use thereof

ABSTRACT

The present disclosure provides antibodies and antibody fragments thereof that bind to IL-23p19. The disclosed antibodies and antibody fragments thereof can modulate a biological activity of the IL-23 receptor signaling axis and are therefore useful for the treatment of immune-mediated inflammatory disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) from U.S.Provisional Application Ser. No. 62/951,231, filed Dec. 20, 2019, all ofwhich is hereby incorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 16, 2019, isnamed “122863-5002-US_NVRB-004-001_ST25.TXT” and is 13 kilobytes insize.

FIELD

The present disclosure generally relates to antibodies and antibodyfragments thereof that bind the p19 subunit of interleukin-23. Theantibodies are useful for the treatment of immune-mediated inflammatorydisorders, autoimmune diseases, or cancer.

BACKGROUND

The interleukin-12 (IL-12) family of regulatory cytokines includes aunique group of cytokines (IL-12, IL-23, IL-27, IL-35, and IL-39)comprising covalently bonded heterodimeric subunits. The heterodimericIL-12 family cytokine members consist of an α-chain (p19, p28 or p35)and a β-chain (p40 or Ebi3).

IL-23 is a heterodimeric cytokine comprising a unique p19 subunit linkedwith a p40 subunit which is shared with IL-12. The main sources of IL-23are tissue-resident or recruited dendritic cells and macrophages. Thebiologic action of IL-23 is hypothesized to occur through a receptorcomplex which is composed of the following two parts: i.) IL-12Rβ1, apart in common with IL-12, and ii.) IL-23R, a part specific for IL-23.

Members of the IL-12 family of cytokines act as immunological playmakersby directing innate and adaptive immune responses. These regulatorycytokines act by inducing the development of T-cell subpopulations andaltering the function and fate of many immune cell populations thatdirect adaptive immune responses to infection, inflammation andautoimmune disease outcomes. IL-12 and IL-23 are predominantlyproinflammatory/prostimulatory cytokines that play roles in thedevelopment of Th1 and Th17 cells, respectively.

The functional IL-23 receptor is a heterodimer of the IL-12Rβ1 subunit,which is shared with the IL-12 receptor and partnered with the signalingchain IL-23R (p19 subunit binding). The receptor for IL-23 isconstitutively associated with Janus kinase 2 (Jak2) and predominantlyactivates STAT3. Expression of the IL-23 receptor is detected primarilyon memory T-cells and NK cells. Monocytes, macrophages and dendriticcells also express IL-23 receptor at low levels.

There is substantial evidence that IL-23 responsive cells are associatedwith autoimmune inflammatory diseases and cancer and that the modulationof IL-23 activity can provide promising therapies. In particular,abnormal regulation of IL-23 is associated with immune-mediatedinflammatory diseases (IMIDs), such as psoriasis, psoriatic arthritis,Crohn's disease and ulcerative colitis. In addition, the balance ofproinflammatory cytokines, including IL-23 and IL-12 plays a key role inshaping the development of antitumor or protumor immunity.

The IL-23/IL-12 pathways are implicated in the cellular mechanismsinvolved in the pathophysiology of multiple inflammatory diseases.Several therapeutic strategies have been designed to inhibit IL-23activity and there is a continuing need for therapeutic agents thattarget the pro-inflammatory IL-23/IL-23 receptor signaling axis fortreatment of immune-mediated inflammatory disorders. More specifically,there remains a need for selective IL-23p19 antagonist antibodies thatbind with high affinity to the p19 subunit of IL-23, in particular,human IL-23, and do not bind to the p40 subunit of the related cytokinefamily member, IL-12.

SUMMARY

The present disclosure addresses the above need by providing antibodiesand antibody fragments that bind to the cytokine p19 subunit of IL-23.The antibodies and antibody fragments are useful for the treatment ofimmune-mediated inflammatory diseases (IMIDs) (e.g., autoimmune diseasesand inflammatory disorders), either alone (e.g., as a monotherapy) or incombination with other immunotherapeutic agents.

In some embodiments, the anti-IL-23p19 antibodies or antibody fragmentsthereof bind to the cytokine p19 subunit of human IL-23. In a furtherembodiment, the antibodies are fully human.

In some embodiments, the anti-IL-23p19 antibodies or antibody fragmentsthereof comprise a heavy chain variable region comprising CDR1: SEQ IDNO: 9, CDR2: SEQ ID NO: 10, and CDR3: SEQ ID NO: 11; and/or a lightchain variable region comprising CDR1: SEQ ID NO: 12, CDR2: SEQ ID NO:13, and CDR3: SEQ ID NO: 14.

In some embodiments, the anti-IL-23p19 antibodies or antibody fragmentsthereof comprise a heavy chain variable region comprising CDR1: SEQ IDNO: 15, CDR2: SEQ ID NO: 16, and CDR3: SEQ ID NO: 17, and/or a lightchain variable region comprising CDR1: SEQ ID NO: 18, CDR2: SEQ ID NO:19, and CDR3: SEQ ID NO: 20.

In some embodiments, the anti-IL-23p19 antibodies or antibody fragmentsthereof comprise a heavy chain variable region comprising CDR1: SEQ IDNO: 21, CDR2: SEQ ID NO: 22, and CDR3: SEQ ID NO: 23; and/or a lightchain variable region comprising CDR1: SEQ ID NO: 24, CDR2: SEQ ID NO:25, and CDR3: SEQ ID NO: 26.

In some embodiments, the anti-IL-23p19 antibodies or antibody fragmentsthereof comprise a heavy chain variable region comprising CDR1: SEQ IDNO: 27, CDR2: SEQ ID NO: 28, and CDR3: SEQ ID NO: 29; and/or a lightchain variable region comprising CDR1: SEQ ID NO: 30, CDR2: SEQ ID NO:31, and CDR3: SEQ ID NO: 32.

In some embodiments, the anti-IL-23p19 antibodies or antibody fragmentsthereof comprise a variable heavy chain sequence selected from the groupconsisting of SEQ ID NOs: 1, 3, 5, and 7.

In other embodiments, the anti-IL-23p19 antibodies or antibody fragmentsthereof comprise a variable light chain sequence selected from the groupconsisting of SEQ ID NOs: 2, 4, 6, and 8.

In other embodiments, the anti-IL-23p19 antibodies or antibody fragmentsthereof comprise a variable heavy chain sequence selected from the groupconsisting of SEQ ID NOs: 1, 3, 5, and 7, and a variable light chainsequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and8.

In some embodiments, the anti-IL-23p19 antibody or antibody fragmentcomprises variable heavy chain and variable light chain sequences,selected from the following combinations:

-   -   (a) a variable heavy chain sequence comprising SEQ ID NO: 1 and        a variable light chain sequence comprising SEQ ID NO: 2;    -   (b) a variable heavy chain sequence comprising SEQ ID NO: 3 and        a variable light chain sequence comprising SEQ ID NO: 4;    -   (c) a variable heavy chain sequence comprising SEQ ID NO: 5 and        a variable light chain sequence comprising SEQ ID NO: 6; and    -   (d) a variable heavy chain sequence comprising SEQ ID NO: 7 and        a variable light chain sequence comprising SEQ ID NO: 8.

In some embodiments, the anti-IL-23p19 antibodies (e.g., antagonistantibodies) bind with high affinity to the p19 subunit of IL-23 and donot bind to the p40 subunit of the related cytokine family member,IL-12.

In some embodiments, the anti-IL-23p19 antibodies or antibody fragmentsthereof exhibit one or more of the following characteristics: (a) isspecific for human IL-23p19 and has the ability to block IL-23 bindingto its receptor (IL-23R); (b) inhibits, interferes with, or modulatesIL-23p19 interaction with IL-23 receptor signal transduction; (c)inhibits STAT3 activation induced by IL-23 in DB cells; (d) inhibitsIL-17 production induced by human IL-23 in mouse splenocytes; (e)inhibits IL-17 production induced by human IL-23 in activated humanPBMC; (f) does not inhibit IL-23 interaction with IL-12Rβ1 signaltransduction: (g) does not inhibit human IL-12 induced interferon gammaproduction in human activated T-cells (PBMC) (h) does not inhibitcynomolgus monkey IL-12 induced interferon gamma production in humanactivated T-cells (PBMC); and (i) inhibits skin inflammation induced byhuman IL-23 in a murine psoriasis-like model.

In one aspect, the disclosed antibodies and isolated antigen bindingagents can be used to inhibit IL-23p19 induced IL-23 receptor signalingnetworks (e.g., of the inflammatory microenvironment that promoteautoimmune diseases).

The anti-IL-23p19 antibodies or antibody fragments thereof may exhibitone or more of the following properties:

-   -   (a) is specific for human IL-23p19 and has the ability to block        IL-23 binding to its receptor IL-23 receptor (e.g., blocker);    -   (b) inhibits, interferes with, or modulates IL-23/IL-23        receptor-mediated signal transduction;    -   (c) blocks IL-23-induced STAT3 activation induced by IL-23 in DB        cells;    -   (d) inhibits IL-23-induced IL-17 production in mouse        splenocytes;    -   (e) inhibits IL-23-induced IL-17 production in human PBMC;    -   (f) does not inhibit IL-23 interaction with IL-12Rβ1 signal        transduction;    -   (g) does not block human IL-12-induced interferon-7 production        in human PBMC;    -   (h) does not inhibit cynomolgus monkey IL-12-induced interferon        gamma productions in human PBMC; and    -   (i) inhibits IL-23-induced skin-inflammation in a murine        psoriasis-like model.

In some embodiments, the anti-IL-23p19 antibodies or antibody fragmentsthereof comprise a combination of CDR sequences derived from a variableheavy chain sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, and 7, and a variable light chain sequence selected from thegroup consisting of SEQ ID NOs: 2, 4, 6, and 8.

In some embodiments, the anti-IL-23p19 antibodies and antibody fragmentsthereof comprise one or more heavy chain variable region CDRs disclosedin Table 1 and/or one or more light chain variable region CDRs disclosedin Table 2.

In some embodiments, the anti-IL-23p19 antibody or antibody fragment isa recombinant antibody (e.g., a chimeric antibody or a humanizedantibody) and comprises six (6) CDRs, all derived from the VH or VLdomain of a single anti-IL-23p19 antibody disclosed herein. For example,a binding agent may comprise all six of the CDR regions of theanti-IL-23p19 antibody designated Hu-2.18006B (for a human antibody). Ina representative example an antibody or antibody fragment thereof maycomprise the amino acid sequences of SEQ ID NOs: 9-11 and SEQ ID NOs:12-14, representing the CDR1, CDR2 and CDR3 of the variable heavy chainregion and the CDR1, CDR2 and CDR3 of the variable light chain region ofthe Hu-2.18006B antibody.

In some embodiments, the anti-IL-23p19 antibody is a full-lengthantibody.

In some embodiments, the anti-IL-23p19 antibody is an antibody fragment.In further embodiments, the antibody fragment is selected from the groupconsisting of: Fab, Fab′, F(ab′)2, Fd, Fv, scFv and scFv-Fc fragment, asingle-chain antibody, a minibody, and a diabody.

In some embodiments, the anti-IL-23p19 antibody is a monoclonalantibody.

In some embodiments, the anti-IL-23p19 antibody is a human antibody. Insome embodiments, the anti-IL-23p19 antibody is a murine antibody.

In some embodiments, the anti-IL-23p19 antibody is a chimeric antibody.In some embodiments, the anti-IL-23p19 antibody is a bispecificantibody. In some embodiments, the anti-IL-23p19 antibody is a humanizedantibody.

The anti-IL-23p19 antibodies and antibody fragments thereof may be usedfor the treatment or prevention of an immune-mediated inflammatorydisease (IMID), such as an autoimmune diseases or inflammatorydisorders, or cancer. Such methods for the treatment or prevention of anIMID or cancer comprise administering a composition or formulation thatcomprises an anti-IL-23p19 antibody or antibody fragment thereof to asubject in need thereof. In a further embodiment, the anti-IL-23p19antibody or antibody fragment thereof may be administered either alone(e.g., as a monotherapy) or in combination with other immunotherapeuticagent and/or a chemotherapy. The IMID may be selected from the groupconsisting of, psoriasis, psoriatic arthritis, inflammatory boweldiseases (e.g., ulcerative colitis or Crohn's disease) ankylosingspondylitis, systemic lupus erythematosus, hidradenitis suppurativa,atopic dermatitis, asthma and familial adenomatous polyposis (FAP).

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe disclosure, will be better understood when read in conjunction withthe appended figures. For the purpose of illustrating the disclosure,shown in the figures are embodiments which are presently preferred. Itshould be understood, however, that the disclosure is not limited to theprecise arrangements, examples and instrumentalities shown.

FIGS. 1A-1D provide the amino acid sequences of the VH and VL domains ofthe anti-IL-23p19 antibodies and their respective CDR sequences.Sequence identifiers are provided and the CDRs are underlined in thecontext of the variable domain sequence.

FIGS. 2A, 2B, 2C, 2D and 2E show the binding profiles of theanti-IL-23p19 antibodies to human IL-23, a recombinant cytokinecomprising human p19 and murine p40 subunits, human IL-12 and human p40subunit, determined by BIAcore.

FIGS. 3A, 3B and 3C show dose-dependent binding of the selectedrepresentative IL-23p19 antibodies to human IL-23 and a recombinantcytokine comprising human p19 and murine p40 subunits determined byELISA.

FIGS. 3D and 3E show no binding of the selected representativeanti-IL-23p19 antibodies to human IL-12 and human p40 subunit determinedby ELISA.

FIG. 4 shows blocking of the IL-23/IL-23 receptor interaction by thefour IL-23p19 antibodies as determined by ELISA.

FIG. 5 shows two representative IL-23p19 antibodies that do not blockIL-23/IL-12 receptor β1 interaction.

FIGS. 6A and 6B show inhibition of IL-23-induced IL-17 production bythree representative anti-IL-23p19 antibodies in a mouse splenocyteassay (MSA).

FIG. 7 shows inhibition of IL-23-induced STAT3 activation in a reportercell assay by two representative anti-IL-23p19 antibodies.

FIG. 8 shows no inhibition of human IL-12-induced IFN-γ production inhuman PBMCs by two representative anti-IL-23p19 specific antibodies.

FIG. 9 shows no inhibition of cynomolgus monkey IL-12-induced IFN-γproduction in human PBMCs by two representative anti-IL-23p19antibodies.

FIG. 10 shows in vivo inhibition of an IL-23 mediated inflammatoryresponse (thickness of the ears) by two representative anti-IL-23p19antibodies in a murine skin inflammation model explained in Example 7.

FIGS. 11A, 11B, 11C and 11D provide graphics of the representation ofthe pathology scores (H&E staining of the frozen ear tissues) effectsfrom two anti-IL-23p19 antibodies on day 8 after the treatment from themice treated in the murine skin inflammation model presented in Example7.

FIGS. 12A, 12B, 12C and 12D shows representative photos of hematoxylinand eosin (H&E) staining of frozen ear tissues collected on the last dayof the in vivo study (day 8) from the mice treated in the murine skininflammation model presented in Example 7.

DETAILED DESCRIPTION

IL-23 is a pro-inflammatory heterodimeric cytokine that comprises a p19submit and binds to an IL-23 receptor. Targeting the pro-inflammatoryIL-23/IL-23 receptor signaling axis is an area of intense therapeuticexploration. The present disclosure provides antibodies and antibodyfragments thereof that inhibit human IL-23/IL-23 receptor signaling axisand can be used for the treatment or prevention of IMIDs.Advantageously, the anti-IL-23p19 antibodies disclosed herein allow forcomplete inhibition of IL-23p19, result in lower dose formulations,result in less frequent and/or more effective dosing, and lead toreduced cost and increased efficiency.

The anti-IL-23p19 antibodies and antibody fragments thereof disclosedherein specifically bind to human IL-23p19 and antagonize theIL-23/IL-23 receptor signaling axis. In one aspect, the disclosedantibodies and antibody fragments thereof bind to human IL-23 with highaffinity and prevent its interaction with the IL-23R, thereby blockingthe downstream signaling cascade. In a particular aspect, the antibodiesor antibody fragments thereof inhibit the IL-23 stimulated production ofIL-17 from mouse splenocytes and from human PBMC. In another aspect, theantibodies or antibody fragments thereof do not bind to nor antagonizeIL-12.

So that the disclosure may be more readily understood, certain technicaland scientific terms are specifically defined below. Unless specificallydefined elsewhere in this document, all other technical and scientificterms used herein have the meaning commonly understood by one ofordinary skill in the art to which this disclosure belongs.

Throughout this disclosure the following abbreviations will be used:

mAb or Mab or MAb—Monoclonal antibody.

CDR—Complementarity determining region in the immunoglobulin variableregions.

VH or VH—Immunoglobulin heavy chain variable region.

VL or VL—Immunoglobulin light chain variable region.

FR—Antibody framework region, the immunoglobulin variable regionsexcluding the CDR regions

As used herein the term “interleukin-23” (used interchangeably withIL-23) refers to the human IL-23 heterodimer including, for example, ahuman IL-23 heterodimer comprising or consisting of a protein subunithaving the amino acid sequence provided in UniProt entryUniProtKB-P29460 identified as IL-23 subunit (p40) disulfide-linked to aprotein subunit having the amino acid sequence provided in UniProt entryUniProtKB-Q9NPF7 identified as Interleukin-23 subunit alpha (p19).

As used herein the term “IL-12R complex” and “IL-12R” refers to thehigh-affinity IL-12 cytokine receptor complex comprising the IL-12Rβ1and IL-12Rβ2 subunits.

As used herein the term “IL-23R complex” and “IL-23R refers to thehigh-affinity IL-23 cytokine receptor comprising the IL-12Rβ1 (in commonwith the IL-12R complex) and IL-23R subunits.

As used herein the term “interleukin-12” (used interchangeablythroughout this disclosure with IL-12) refers to the human IL-12heterodimer including, for example, a human IL-12 heterodimer comprisingor consisting of a protein subunit having the amino acid sequenceprovided in UniProt entry UniProtKB-P29459 (identified as interleukin-12subunit alpha disulfide-linked to a protein subunit comprising the aminoacid sequence provided in UniProt entry UniProtKB-P29460 (identified asinterleukin-12 subunit beta) (p40)). The term includes a heterodimericprotein comprising a 35 kD subunit (p35) and a 40 kD subunit (p40) whichare both linked together with a disulfide bridge. The heterodimericprotein is referred to as a “p70 subunit”. The structure of human IL-12is described further in, for example, Kobayashi, et al. (1989) J. ExpMed. 170:827-845 and Ling, et al. (1995) J. Exp Med. 154:116-127). Theterm human IL-12 is intended to include recombinant human IL-12 (rhIL-12), which can be prepared by standard recombinant expressionmethods.

As used herein the term “interleukin 17” also referred to as “IL-17” or“IL-17A” is a 20-30 kD glycosylated homodimeric protein including, forexample, a homodimeric protein comprising or consisting of a proteinsubunit having the amino acid sequence provided in UniProt entryUniProtKB-Q16552. The human IL-17 gene codes for a 155 amino acidprotein that has a 19 amino acid signal sequence and a 136 amino acidmature segment. IL-17 is secreted by activated T-cells at sites ofinflammation but is typically not present in the systemic circulation.IL-17 binds to a type I transmembrane receptor termed IL-17R which is alarge ubiquitously expressed protein that demonstrates no significantsequence similarity to other known cytokine receptors. Human IL-17 showsamino acid sequence identity of 62.5% and 58% to the mouse and rat aminoacid IL-17 sequences, respectively. Human IL-17 shows amino acidsequence identity of 97.4% to the cynomolgus monkey IL-17.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, and multispecific antibodies (e.g.,bispecific antibodies).

An exemplary antibody such as an IgG comprises two heavy chains and twolight chains. Each heavy chain is comprised of a heavy chain variableregion (abbreviated herein as VH) and a heavy chain constant region.Each light chain is comprised of a light chain variable region(abbreviated herein as VL) and a light chain constant region. The VH andVL regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). Each VHand VL is composed of three CDRs and four FRs, arranged from aminoterminus to carboxy-terminus in the following order: FR1, CDR1, FR2,CDR2, FR3, CDR3, FR4.

The hypervariable region generally encompasses amino acid residues fromabout amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56(LCDR2) and 89-97 (LCDR3) in the light chain variable region and aroundabout 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102(HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991) and/or thoseresidues forming a hypervariable loop (e.g. residues 26-32 (LCDR1),50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chainvariable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,e.g., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies and is not to be construed as requiringproduction of the antibody by any method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by a variety of techniques, including but not limited to thehybridoma method, recombinant DNA methods, phage-display methods, andmethods utilizing transgenic animals containing all or part of the humanimmunoglobulin loci, such methods and other exemplary methods for makingmonoclonal antibodies being described herein.

The term “chimeric” antibody refers to a recombinant antibody in which aportion of the heavy and/or light chain is derived from a particularsource or species, while the remainder of the heavy and/or light chainis derived from a different source or species.

A “human antibody” is an antibody that possesses an amino-acid sequencecorresponding to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies known toone of skill in the art. This definition of a human antibodyspecifically excludes a humanized antibody comprising non-humanantigen-binding residues. Human antibodies can be produced using varioustechniques known in the art, including methods described in Cole et al,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985);Boerner et al, J. Immunol, 147(I):86-95 (1991). See also van Dijk andvan de Winkel, Curr. Opin. Pharmacol, 5: 368-74 (2001). Human antibodiescan be prepared by administering the antigen to a transgenic animal thathas been modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled, e.g., immunizedHuMab mice (see, e.g., Nils Lonberg et al., 1994, Nature 368:856-859, WO98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO01/09187 regarding HuMab mice), xenomice (see, e.g., U.S. Pat. Nos.6,075,181 and 6,150,584 regarding XENOMOUSE™ technology) or Trianni mice(see, e.g., WO 2013/063391, WO 2017/035252 and WO 2017/136734).

The term “humanized antibody” refers to an antibody that has beenengineered to comprise one or more human framework regions in thevariable region together with non-human (e.g., mouse, rat, or hamster)complementarity-determining regions (CDRs) of the heavy and/or lightchain. In certain embodiments, a humanized antibody comprises sequencesthat are entirely human except for the CDR regions. Humanized antibodiesare typically less immunogenic to humans, relative to non-humanizedantibodies, and thus offer therapeutic benefits in certain situations.Those skilled in the art will be aware of humanized antibodies and willalso be aware of suitable techniques for their generation. See forexample, Hwang, W. Y. K., et al., Methods 36:35, 2005; Queen et al.,Proc. Natl. Acad. Sci. USA, 86:10029-10033, 1989; Jones et al., Nature,321:522-25, 1986; Riechmann et al., Nature, 332:323-27, 1988; Verhoeyenet al., Science, 239:1534-36, 1988; Orlandi et al., Proc. Natl. Acad.Sci. USA, 86:3833-37, 1989; U.S. Pat. Nos. 5,225,539; 5,530,101;5,585,089; 5,693,761; 5,693,762; 6,180,370; and Selick et al., WO90/07861, each of which is incorporated herein by reference in itsentirety.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG1, IgG2,IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The terms “antigen-binding domain” of an antibody (or simply “bindingdomain”) of an antibody or similar terms refer to one or more fragmentsof an antibody that retain the ability to specifically bind to anantigen complex. Examples of binding fragments encompassed within theterm “antigen-binding portion” of an antibody include (i) Fab fragments,monovalent fragments consisting of the VL, VH, CL and CH domains; (ii)F(ab′)2 fragments, bivalent fragments comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) Fd fragmentsconsisting of the VH and CH domains; (iv) Fv fragments consisting of theVL and VH domains of a single arm of an antibody, (v) dAb fragments(Ward et al., (1989) Nature 341: 544-546), which consist of a VH domain;(vi) isolated complementarity determining regions (CDR), and (vii)combinations of two or more isolated CDRs which may optionally be joinedby a synthetic linker.

“Complementarity determining region” or “CDR” as the terms are usedherein refer to short polypeptide sequences within the variable regionof both heavy and light chain polypeptides that are primarilyresponsible for mediating specific antigen recognition. There are threeCDRs (termed CDR1, CDR2, and CDR3) within each VL and each VH.

As will be appreciated by those in the art, the exact numbering andplacement of the CDRs can be different among different numberingsystems. However, it should be understood that the disclosure of avariable heavy and/or variable light sequence includes the disclosure ofthe associated CDRs. Accordingly, the disclosure of each variable heavyregion is a disclosure of the vhCDRs (e.g. vhCDR1, vhCDR2 and vhCDR3)and the disclosure of each variable light region is a disclosure of thevlCDRs (e.g. vlCDR1, vlCDR2 and vlCDR3).

In certain embodiments, the CDRs of an antibody can be determinedaccording to the IMGT numbering system as described in Lefranc M-P,(1999) The Immunologist 7: 132-136 and Lefranc M-P et al, (1999)NucleicAcids Res 27: 209-212, each of which is herein incorporated by referencein its entirety. Unless stated otherwise herein, references to residuenumbers in the variable domain of antibodies means residue numbering bythe IMGT numbering system.

In other embodiments, the CDRs of an antibody can be determinedaccording to MacCallum R M et al, (1996) J Mol Biol 262: 732-745, hereinincorporated by reference in its entirety. See also, e.g. Martin A.“Protein Sequence and Structure Analysis of Antibody Variable Domains,”in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp.422-439, Springer-Verlag, Berlin (2001), herein incorporated byreference in its entirety. In other embodiments, the CDRs of an antibodycan be determined according to the AbM numbering scheme, which refers toAbM hypervariable regions, which represent a compromise between theKabat CDRs and Chothia structural loops, and are used by OxfordMolecular's AbM antibody modeling software (Oxford Molecular Group,Inc.), herein incorporated by reference in its entirety.

“Framework” or “framework region” or “FR” refers to variable domainresidues other than hypervariable region (HVR) residues. The FR of avariable domain generally consists of four FR domains: FR1, FR2, FR3,and FR4.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), Vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup Illas in Kabat et al., supra.

The “hinge region” is generally defined as stretching from 216-238 (EUnumbering) or 226-251 (Kabat numbering) of human IgG1. The hinge can befurther divided into three distinct regions, the upper, middle (e.g.,core), and lower hinge.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991).

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds. Certain blockingantibodies or antagonist antibodies substantially or completely inhibitthe biological activity of the antigen.

The term “Effector functions” refer to those biological activitiesattributable to the Fc region of an antibody, which vary with theantibody isotype. Examples of antibody effector functions include: C1qbinding and complement dependent cytotoxicity (CDC); Fc receptorbinding; antibody-dependent T-cell-mediated cytotoxicity (ADCC);phagocytosis; down-regulation of cell surface receptors (e.g., B-cellreceptor); and B-cell activation.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that contacts an overlapping set of amino acidresidues of the antigen as compared to the reference antibody or blocksbinding of the reference antibody to its antigen in a competition assayby 50% or more. The amino acid residues of an antibody that contact anantigen can be determined, for example, by determining the crystalstructure of the antibody in complex with the antigen or by performinghydrogen/deuterium exchange. In some embodiments, residues of anantibody that are within 5 Å the antigen are considered to contact theantigen. In some embodiments, an antibody that binds to the same epitopeas a reference antibody blocks binding of the reference antibody to itsantigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more.

The term “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab)₂;diabodies; linear antibodies; single-chain antibody molecules (e.g.,scFv). Papain digestion of antibodies produces two identicalantigen-binding fragments, called “Fab” fragments, and a residual “Fc”fragment, a designation reflecting the ability to crystallize readily.The Fab fragment consists of an entire light (L) chain along with thevariable region domain of the heavy (H) chain (VH), and the firstconstant domain of one heavy chain (CH1). Pepsin treatment of anantibody yields a single large F(ab)₂ fragment which roughly correspondsto two disulfide linked Fab fragments having divalent antigen-bindingactivity and is still capable of cross-linking antigen. Fab fragmentsdiffer from Fab′ fragments by having additional few residues at thecarboxy terminus of the CH1 domain including one or more cysteines fromthe antibody hinge region. Fab′-SH is the designation herein for Fab′ inwhich the cysteine residue(s) of the constant domains bear a free thiolgroup. F(ab′)₂ antibody fragments originally were produced as pairs ofFab′ fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

“Fv” consists of a dimer of one heavy- and one light-chain variableregion domain in tight, non-covalent association. From the folding ofthese two domains emanate six hypervariable loops (3 loops each from theH and L chain) that contribute the amino acid residues for antigenbinding and confer antigen binding specificity to the antibody.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the VH and VL antibody domains connected into asingle polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the VH and VL domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

The terms “antigen-binding domain” of an antibody (or simply “bindingdomain”) of an antibody or similar terms refer to one or more fragmentsof an antibody that retain the ability to specifically bind to anantigen complex. Examples of binding fragments encompassed within theterm “antigen-binding portion” of an antibody include (i) Fab fragments,monovalent fragments consisting of the VL, VH, CL and CH domains; (ii)F(ab′)₂ fragments, bivalent fragments comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) Fd fragmentsconsisting of the VH and CH domains; (iv) Fv fragments consisting of theVL and VH domains of a single arm of an antibody, (v) dAb fragments(Ward et al., (1989) Nature 341: 544-546), which consist of a VH domain;(vi) isolated complementarity determining regions (CDR), and (vii)combinations of two or more isolated CDRs which may optionally be joinedby a synthetic linker.

The term “multispecific antibody” is used in the broadest sense andspecifically covers an antibody comprising a heavy chain variable domain(VH) and a light chain variable domain (VL), where the VH-VL unit haspolyepitopic specificity (e.g., is capable of binding to two differentepitopes on one biological molecule or each epitope on a differentbiological molecule). Such multispecific antibodies include, but are notlimited to, full-length antibodies, antibodies having two or more VL andVH domains, bispecific diabodies and triabodies. “Polyepitopicspecificity” refers to the ability to specifically bind to two or moredifferent epitopes on the same or different target(s).

“Dual specificity” or “bispecificity” refers to the ability tospecifically bind to two different epitopes on the same or differenttarget(s). However, in contrast to bispecific antibodies, dual-specificantibodies have two antigen-binding arms that are identical in aminoacid sequence and each Fab arm is capable of recognizing two antigens.Dual-specificity allows the antibodies to interact with high affinitywith two different antigens as a single Fab or IgG molecule. Accordingto one embodiment, the multispecific antibody in an IgG1 form binds toeach epitope with an affinity of 5 μM to 0.001 μM, 3 μM to 0.001 μM, 1μM to 0.001 μM, 0.5 μM to 0.001 μM or 0.1 μM to 0.001 μM. “Monospecific”refers to the ability to bind only one epitope. Multi-specificantibodies can have structures similar to full immunoglobulin moleculesand include Fc regions, for example IgG Fc regions. Such structures caninclude, but are not limited to, IgG-Fv, IgG-(scFv)₂, DVD-Ig,(scFv)₂-(scFv)₂-Fc and (scFv)₂-Fc-(scFv)₂. In case of IgG-(scFv)₂, thescFv can be attached to either the N-terminal or the C-terminal end ofeither the heavy chain or the light chain.

As used herein, the term “bispecific antibodies” refers to monoclonal,often human or humanized, antibodies that have binding specificities forat least two different antigens. In the invention, one of the bindingspecificities can be directed towards IL-12 or IL-23, the other can befor any other antigen, e.g., for a cell-surface protein, receptor,receptor subunit, tissue-specific antigen, virally derived protein,virally encoded envelope protein, bacterially derived protein, orbacterial surface protein, etc.

As used herein, the term “diabodies” refers to bivalent antibodiescomprising two polypeptide chains, in which each polypeptide chainincludes VH and VL domains joined by a linker that is too short (e.g., alinker composed of five amino acids) to allow for intramolecularassociation of VH and VL domains on the same peptide chain. Thisconfiguration forces each domain to pair with a complementary domain onanother polypeptide chain so as to form a homodimeric structure.Accordingly, the term “triabodies” refers to trivalent antibodiescomprising three peptide chains, each of which contains one VH domainand one VL domain joined by a linker that is exceedingly short (e.g., alinker composed of 1-2 amino acids) to permit intramolecular associationof VH and VL domains within the same peptide chain.

The term an “isolated antibody” when used to describe the variousantibodies disclosed herein, means an antibody that has been identifiedand separated and/or recovered from a cell or cell culture from which itwas expressed. Contaminant components of its natural environment arematerials that would typically interfere with diagnostic or therapeuticuses for the polypeptide, and can include enzymes, hormones, and otherproteinaceous or non-proteinaceous solutes. In some embodiments, anantibody is purified to greater than 95% or 99% purity as determined by,for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing(IEF), capillary electrophoresis) or chromatographic (e.g., ion exchangeor reverse phase HPLC) approaches. For a review of methods forassessment of antibody purity, see, for example, Flatman et al., J.Chromatogr. B 848:79-87 (2007). In a preferred embodiment, the antibodywill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain.

With regard to the binding of an antibody to a target molecule, the term“specific binding” or “specifically binds to” or is “specific for” aparticular polypeptide or an epitope on a particular polypeptide targetmeans binding that is measurably different from a non-specificinteraction. Specific binding can be measured, for example, bydetermining binding of a molecule compared to binding of a controlmolecule. For example, specific binding can be determined by competitionwith a control molecule that is similar to the target, for example, anexcess of non-labeled target. In this case, specific binding isindicated if the binding of the labeled target to a probe iscompetitively inhibited by excess unlabeled target. The term “specificbinding” or “specifically binds to” or is “specific for” a particularpolypeptide or an epitope on a particular polypeptide target as usedherein can be exhibited, for example, by a molecule having a Kd for thetarget of 10-4 M or lower, alternatively 10-5 M or lower, alternatively10-6 M or lower, alternatively 10-7 M or lower, alternatively 10-8 M orlower, alternatively 10-9 M or lower, alternatively 10-10 M or lower,alternatively 10-11 M or lower, alternatively 10-12 M or lower or a Kdin the range of 10-4 M to 10-6 M or 10-6 M to 10-10 M or 10-7 M to 10-9M. As will be appreciated by the skilled artisan, affinity and KD valuesare inversely related. A high affinity for an antigen is measured by alow KD value. In one embodiment, the term “specific binding” refers tobinding where a molecule binds to a particular polypeptide or epitope ona particular polypeptide without substantially binding to any otherpolypeptide or polypeptide epitope. As used herein the terms “specificbinding,” “specifically binds,” and “selectively binds,” refer toantibody binding to an epitope of a human interleukin-23 p19.

The term “affinity,” as used herein, means the strength of the bindingof an antibody to an epitope. The affinity of an antibody is given bythe dissociation constant Kd, defined as [Ab]×[Ag]/[Ab-Ag], where[Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab]is the molar concentration of the unbound antibody and [Ag] is the molarconcentration of the unbound antigen. The affinity constant Ka isdefined by 1/Kd. Methods for determining the affinity of mAbs can befound in Harlow, et al., Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan etal., eds., Current Protocols in Immunology, Greene Publishing Assoc. andWiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol.92:589-601 (1983), which references are entirely incorporated herein byreference. One standard method well known in the art for determining theaffinity of mAbs is the use of surface plasmon resonance (SPR) screening(such as by analysis with a BIAcore™ SPR analytical device).

An “epitope” is a term of art that indicates the site or sites ofinteraction between an antibody and its antigen(s). As described by(Janeway, C, Jr., P. Travers, et al. (2001). Immunobiology: the immunesystem in health and disease. Part II, Section 3-8. New York, GarlandPublishing, Inc.): “An antibody generally recognizes only a small regionon the surface of a large molecule such as a protein . . . [Certainepitopes] are likely to be composed of amino acids from different partsof the [antigen] polypeptide chain that have been brought together byprotein folding. Antigenic determinants of this kind are known asconformational or discontinuous epitopes because the structurerecognized is composed of segments of the protein that are discontinuousin the amino acid sequence of the antigen but are brought together inthe three-dimensional structure. In contrast, an epitope composed of asingle segment of polypeptide chain is termed a continuous or linearepitope” (Janeway, C. Jr., P. Travers, et al. (2001). Immunobiology: theimmune system in health and disease. Part II, Section 3-8. New York,Garland Publishing, Inc.).

The term “KD”, as used herein, is intended to refer to the dissociationconstant of a particular antibody-antigen interaction. It is calculatedby the formula: Koff/Kon=KD

The term “IC50”, as used herein, is intended to refer to the effectiveconcentration of antibody of the present invention needed to neutralize50% of the bioactivity of IL-23 on human lymphoma DB cells in thebioassay described in Example 5: Inhibition of STAT3 activation in humanDB cell Assay.

“EC50” with respect to an agent and a particular activity (e.g. bindingto a cell, inhibition of enzymatic activity, activation or inhibition ofan immune cell), refers to the efficient concentration of the agentwhich produces 50% of its maximum response or effect with respect tosuch activity. “EC100” with respect to an agent and a particularactivity refers to the efficient concentration of the agent whichproduces its substantially maximum response with respect to suchactivity.

As used herein the term “antibody-based immunotherapy” and“immunotherapy” are used to broadly refer to any form of therapy thatrelies on the targeting specificity of an anti-IL-23p19 antibody,bispecific molecule, multi-specific molecule, binding agent, or fusionprotein comprising an IL-23p19 specific binding agent, to mediate adirect or indirect effect on a cell characterized by aberrant expressionof IL-23p19. The terms are meant to encompass methods of treatment usingnaked antibodies, bispecific antibodies (including T-cell engaging, NKcell engaging and other immune cell/effector cell engaging formats),antibody drug conjugates, cellular therapies using T-cells (CAR-T) or NKcells (CAR-NK) engineered to comprise an IL-23p19-specific chimericantigen receptor, and oncolytic viruses comprising an IL-23p19 specificbinding agent, and gene therapies by delivering the antigen bindingsequences of the anti-IL-23p19 antibodies and express the correspondingantibody fragments in vivo.

As used herein, the term “immune-mediated inflammatory diseases” or“IMIDs” includes a group of seemingly unrelated diseases that sharecommon inflammatory pathways and are triggered by or result in thedysregulation of innate and adaptive immune system functions. Theseconditions include, but are not limited to, psoriasis, rheumatoidarthritis, inflammatory bowel diseases, systemic lupus erythematosus,ankylosing spondylitis, hidradenitis suppurativa, atopic dermatitis andasthma. Any organ system may be inflicted by an IMID, and individualsmay encounter a considerable reduction in quality of life, significantmorbidity, and reduced lifespan (Bunte, K and Beikler, T, Int. J. Mol.Sci., 20: 3394 (2019)). It is noted here that, as used in thisspecification and the appended claims, the singular forms “a,” “an,” and“the” include plural reference unless the context clearly dictatesotherwise.

IL-12/IL-23 Receptor Signaling Axis

The p19 subunit of IL-23 (also referred to herein as “IL-23p19” and “p19subunit”) is a 189 amino acid polypeptide containing a 21 amino acidleader sequence (Oppmann et al. Immunity 13:715 (2000)). The biologicalactivity of the p19 subunit is only detected when it is partnered withthe IL-12 p40 subunit to form IL-23. Both IL-12 and IL-23 exist only assecreted heterodimeric cytokines and neither the IL-12 p35 nor theIL-23p19 subunit are secreted without intracellular covalent associationwith p40. The p40 subunit shared by the IL-12 and IL-23 cytokines bindthe common IL-12Rβ1 component of their receptors, with signalingspecificity being determined by the unique p35 (Il-12) and p19 (IL-23)subunits that bind the IL-12Rβ2 and IL-23R components of theirrespective high-affinity receptors. The interactions of IL-12 and IL-23with their cognate receptors form part of a complicated regulatorynetwork that coordinates innate and adaptive immune responses.

It is hypothesized that IL-12 plays a critical role in the developmentof protective immune responses to many intracellular pathogens andviruses and in tumor immune surveillance. See Kastelein, et al., AnnualReview of Immunology, 2007, 25: 221-42; Liu, et al., Rheumatology, 2007,46(8): 1266-73; Bowman et al., Current Opinion in Infectious Diseases,2006 19:245-52; Fieschi and Casanova, Eur. J. Immunol. 2003 33:1461-4;Meeran et al., Mol. Cancer. Ther. 2006 5: 825-32; Langowski et al.,Nature 2006 442: 461-5. As such, IL-23 specific inhibition (sparingIL-12 or the shared p40 subunit) may have a potentially superior safetyprofile compared to dual inhibition of IL-12 and IL-23.

The receptor for IL-23 comprises the IL-12Rβ1 subunit, shared in commonwith the IL-12 receptor, partnered with a unique subunit called IL-23R(Parham et al. J. Immunol. 168:5699 (2002)). It has been reported thatIL-23R binds to IL-23 with a high affinity (KD=44±3 nM). In contrast,IL-23 binds to IL-12Rβ1 subunit with a lower affinity (KD=2±1 uM).Binding of IL-23R to IL-23 facilitates IL-12Rβ1 binding to IL-23 with avery high affinity (KD=25±5 nM) (Bloch et al, Immunity, 48, 45-58(2018). The IL-23R is expressed by a great range of cells (naturalkiller cells, macrophages, dendritic cells, memory T-cells,keratinocytes). IL-23 production induces expression of IL-23R creating apositive feedback loop that enhances IL-23 expression.

IL-23 is produced by activated antigen presenting cells and binds toIL-23 receptor complexes expressed on NK cells and T-cells. IL-23, aloneor in combination with other cytokines (e.g., IL-1β), has been shown topromote the production of IL-17A, IL-17F, IL-6 and tumor necrosis factorα (TNFα), which are proinflammatory cytokines known to contribute toinflammatory responses in IMID disorders.

Binding of IL-23p19 to IL-23R results in a restructuring process of theIL-23p19 helical domain, which enables binding of IL-12 p40 to IL-12Rβ1(Bloch, Y et al. Immunity. 2018; 48(1):45-58). This process activatesJAK2 and TYK2, leading to STAT3 and STAT4 formation, which ultimatelyfunction as transcription factors (Parham, C. et al., Immunol.168(11):5699-5708 (2002). IL-23 is a key player in the late stage ofdifferentiation of naive CD4+ T-cells into Th17 cells (Gaffen, S L etal., Nat Rev Immunol. 14(9):585-600 (2014). Being devoid of IL-23R,naive T-cells require other cytokines, such as transforming growthfactor (TGF)-β and IL-6, to modulate the early stage of differentiation.These cytokines induce expression of retinoic acid receptor-relatedorphan receptor-γt as the transcription factor, which promotesexpression of IL-23R. Immature Th17 cells induced by TGF-β and IL-6require exposure to IL-23 to attain pathogenicity. Once matured, Th17cells are capable of producing IL-17 and TNF-α (Kashani, A et al.,Gastroenterology & Hepatology 15(5):255-265 (2019).

Despite the structural similarity between the two cytokines, thebiological activities/functions of IL-23 are distinct from those ofIL-12. IL-23 supports the differentiation and maintenance of naive CD4+T-cells into a novel subset of cells called Th17 cells, which aredistinct from the classical Th1 and Th2 cells. Th17 cells produceinterleukin-17A (IL-17A) and interleukin-17F (IL-17F). Th17 cellsproduce a range of other factors known to drive inflammatory responses,including tumor necrosis factors known to drive inflammatory responses,including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6),granulocyte-macrophage colony-stimulating factor (GM-CSF), CXCL1 andCCL20. NK cells and innate lymphoid cells such as lymphoid tissue induce(LTi)-like cells express IL-23 receptor and retinoic-acid-related orphanreceptor (ROR) gamma and produce IL-17 in response to IL-23. IL-1β andIL-23 also co-stimulate gamma-delta T-cells to induce IL-17 productionwithout T-cell receptor engagement.

Significantly, IL-23 maintains the differentiation and expansion ofnaïve T-cells into the distinct Th17 cell lineage. In the absence ofIL-23, the Th17 phenotype is lost. IL-23 has been described as the“master regulator” of the immune-inflammatory response in IMIDs due toits critical role in maintaining the cytotoxic Th17 cells that producepro-inflammatory cytokine profile. The pathogenicity of IL-23 depends inpart on the dysregulated production of IL-17A, IL-17F and IL-22,providing a rationale for targeting the IL-23/IL-23R axis forimmunotherapy.

Targeting the Pro-Inflammatory IL-23/IL-23 Receptor Signaling Axis

Anti-IL-12/IL-23 antibodies that have been reported to confer atherapeutic benefit in vivo include antibodies ustekinumab (CNTO1275)and briakinumab (ABT-874). Both antibodies target the common IL-12 p40subunit in a region of the p40 subunit that is crucial for IL-12Rβ1binding (Clarke, A. et al. mAbs 2(5):539-549 (2010).

Anti-IL-23 selective antibodies that have reported to confer atherapeutic benefit in vivo include guselkumab (TREMFYA®), tildrakizumab(ILUMYA®), risankizumab (SKYRIZI®), brazikumab (MEDI2070) andmirakizumab (Ly3074828); all of which are specific for the p19 subunitof IL-23. Data from randomized, placebo- and active-controlled phase 3clinical trials show tildrakizumab, guselkumab and risankizumab to havea favorable risk-benefit profile in patients with moderate to severepsoriasis. No significant safety concerns have been observed for any ofthese IL-23p19 inhibitors.

Th1 cells driven by IL-12 were previously thought to be the pathogenicT-cell subset in many autoimmune diseases, however, more recent animalstudies in models of inflammatory bowel disease, psoriasis, inflammatoryarthritis and multiple sclerosis, in which the individual contributionsof IL-12 versus IL-23 were evaluated have established that IL-23, notIL-12, is the key driver in autoimmune/inflammatory disease (Ahern etal., Immun. Rev. 226:147-159 (2008); Cua et al., Nature 421:744-748(2003); Yago et al., Arthritis Res and Ther. 9(5): R96 (2007).

The role of IL-23 in immune-mediated inflammatory responses is alsosupported by genetic studies. Genome-wide association study (GWAS)linked IL-23R polymorphisms with predisposition to autoimmune conditionssuch as psoriasis and psoriatic arthritis (Liu et al., PLoS Genet.4(3)e1000041 (2008), Reveille, et al., Nat. Genet. 42(2): 123-127(2010), and Duerr et al., Science 314(5804):1461-1463 (2006). Anassociation between rs11209026, a single-nucleotide polymorphism (SNP)in the IL-23R gene, and CD has been established (Reveille, J D et al.).This variant is shown to be protective against CD and UC. The protectivecharacteristic of rs11209026 was confirmed in a meta-analysis thatshowed that carriage of this SNP variant reduced disease risk in acohort of more than 75,000 cases and controls (Jostins, L. Nature491(7422):119-124 (2012). This SNP variant, along with a few othercoding variants of IL-23R, leads to a decrease in the expression ofIL-23R, thus reducing the immune responses mediated through the IL-23axis (J Biol Chem. 291(16):8673-8685 (2016).

While cytokines such as IL-6 and TGF-β1 can promote the differentiationof RORγt+Th17 cells from naïve CD4+ T-cells, IL-23 is required for thefull inflammatory function of these cells. In addition, the binding ofIL-23 to its receptor on activated RORγt+Th17 cells induces furtherexpression of the IL-23 receptor (IL-23R), thus providing a feed-forwardloop for the maintenance and propagation of these cells (Singh, S, etal., MAbs 7(4):1493-1503 (2015).

There is strong evidence that the IL-23/IL-17 axis plays an importantrole in the development of chronic inflammation, and genetic studieshave revealed a potential link between the IL-23 receptor (IL-23R) orits ligand and several inflammatory diseases, including psoriasis,inflammatory bowel disease, and graft-versus-host disease. Targeting theIL-23/IL-17 axis is an area of intense therapeutic exploration in IMIDs,including psoriasis, psoriatic arthritis, inflammatory bowel diseases(ulcerative colitis and Crohns' disease), ankylosing spondylitis, andsystemic lupus erythematosus (SLE).

Generally speaking, IL-23 specific antibodies, such as guselkumab,tildrakizumab, risankizumab, brazikumab or mirakizumab, selectivelybinds to IL-23p19 and inhibit binding of IL-23 to its receptor; therebyantagonizing the action of IL-23 to induce and sustain T helper (Th) 17cells, innate lymphoid cells, γδT-cells, and natural killer (NK) cellsresponsible for tissue inflammation, destruction and/or aberrant tissuerepair associated with an IMID.

Plaque psoriasis or psoriasis (PsO) is a chronic inflammatory,T-cell-mediated skin disorder that is characterized by a complexpathophysiology. The incidence of its occurrence in developed countriesis 1-4%. Psoriasis is the most prevalent autoimmune disease in theUnited States where it affects approximately 7.5 million people. Plaquepsoriasis is the most common form of psoriasis, affecting 80% to 90% ofpatients. Although the pathogenesis of psoriasis is not completelyunderstood, multiple environmental factors, T-cells, dendritic cells,numerous cytokines, and 45 identified gene loci all interact to createthe systemic psoriatic disease state and ultimately psoriatic plaques(Nestle F O, et al., N Engl J Med. 361(5):496-509 (2009), Mahil S K, etal Dermatol Clin. 33(1):1-11 (2015). A synergistic influence of geneticand environmental factors along with the interplay of innate andadaptive immunity eventually leads to the abnormal keratinocyteproliferation and formation of the psoriatic lesions (Chan, J. R, etal., J. Exp. Med, 203(12)2577-2587 (2006).

PsO plaques are typically well-demarcated erythematous, scaly skinlesions characterized by epidermal thickening. Affected keratinocytesactivate dendritic cells, travel to local lymph nodes and releaseseveral cytokines including interleukin IL-12 and IL-23, which activatetype 1 T helper (Th1) and type 17 T helper (Th17) cells, respectively. Tlymphocytes and other cell types release additional cytokines, includingtumor necrosis factor (TNF)-α, IL-22, and IL-17, leading to increasedkeratinocyte activation and the initiation of a self-propelled cycle ofinflammation (Lowes M A et al., Trends Immunol. 34(4):174-81 (2013)).Histologically, there is a marked epidermal hyperplasia accompanied byparakeratosis and a mixed dermal infiltrate, including CD4+ T-cells,dendritic cells, macrophages and mast cells.

Early publications reported the presence of elevated levels of tumornecrosis factor-α α and the p40 subunit of IL-12, accompanied by theoverexpression of IL-12 p40 and IL-23 p40 messenger RNA in psoriaticskin lesions. These findings suggested that the inhibition of IL-12 andIL-23 with a neutralizing antibody to the IL-12/23 p40 subunit proteinmay offer an effective therapeutic approach for the treatment ofpsoriasis (Piskin G, et al., J Immunol 2006, 176: 1908-15). Psoriasiswas initially deemed to be a Th-1 mediated disease (based on thecytokine secretory profile characteristic of T helper 1-type cells:interleukin-2, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ).

The basic role of IL-23 in the pathogenesis of psoriasis has beenclarified, and it is associated with the biology of the Th17 lineage.The initial differentiation of naïve T lymphocytes to Th17 requires thepresence of TGF-β1, IL-6, and IL-1, while IL-23 is necessary for theactivation and maintenance of Th17 in order to secrete thepro-inflammatory cytokines IL-17, IL-22, IL-21, and tumor necrosisfactor-α, which eventually contributes to the formation of the psoriaticskin lesions (Fotaidou, C. et al., Psoriasis: Targets and Therapy 8: 1-5(2018)).

Therefore, although IL-12 and IL-23 are both known to contribute to thedevelopment of Th1 immune responses in psoriasis, IL-23 is nowrecognized as the key driver of Th17 cell differentiation and survival.The primary cytokines produced by Th17 cells are those of thepro-inflammatory IL-17 family, including IL-17A, IL-17B, IL-17C, IL-17D,IL-17E, and IL-17F. IL-17A and IL-17F are similar and bind to the sameIL-17 receptor, a heterodimer comprised of an IL-17RA and an IL-17RCsubunits.

Although early therapeutic strategies targeted Th1 cells as the centralcell type for psoriasis pathogenesis, newer models focus on theIL-23/Th17 axis (Lowes M A, et al. Trends Immunol. 34(4):174-81(2013).The rationale for the new focus is premised on the belief that IL-17 isa key player in the pathogenesis of is psoriasis and the knowledge thatIL-23 drives Th17 cell activation. Moreover, IL-23 stimulates productionof other Th17 cytokines (e.g., IL-22) by other cell types, includinginnate lymphoid type 3 cells and γδ T-cells (Ward, N. L., J InvestigDermatol. 134: 2305-2307 (2014). It has been suggested that inhibitionof IL-23 will block downstream production of IL-17A and IL-22 by Th17cells and that the effect will translate into antagonism of psoriasisimmunopathogenesis.

The IL-23/IL-17 axis is currently considered to be crucial in thepathogenesis of psoriasis and selective IL-23p19 inhibition may bringseveral advantages with respect to IL-12/23 p40 inhibition, or distalblockade of IL-17A or its receptor (Torres, T Drugs 77:1493-1503 (2017).To date, three IL-23p19-subunit specific monoclonal antibodies (i.e.,guselkumab, tildrakizumab and risankizumab) have received approval fromthe United States Food and Drug Administration (FDA) and the EuropeanMedicines Agency (EMA) for the treatment of moderate-to-severe plaquepsoriasis in adults who are candidates for systemic therapy orphototherapy. In July 2020 guselkumab was also approved for thetreatment of adults with active psoriatic arthritis by the FDA.

The high efficacy of IL-23 blockade in psoriasis was demonstrated inearly proof-of-concept testing and phase I clinical trials. A phase 1study showed that a single dose of guselkumab resulted in significantclinical responses in patients with moderate-to-severe plaque psoriasis(Sofen H, et al., J Allergy Clin Immunol; 133:1032-1040 (2014). ThePhase I study also reported that selective antagonism of interleukin-23with guselkumab resulted in clinical improvement of psoriasis,characterized by reductions in epidermal thickness, T-cell anddendritic-cell infiltration, expression of genes associated withpsoriasis, and serum IL-17A levels. The reported finding of a measurableclinical responses in patients with moderate-to-severe psoriasis, aftera single dose of guselkumab supported the emerging theory that selectiveneutralization of IL-23 was a promising therapeutic option.

A rapid onset of guselkumab activity was also observed in a Phase IIdosing study (NCT01483599) evaluating the use of guselkumab at a broadrange of doses and two different dosing intervals for up to 40 weeks ofcontinuous treatment. Efficacy was evident at the earliest assessment(week 4). Several of the guselkumab regimens were associated withconsiderably better response rates than those associated withadalimumab, a biologic agent that is commonly used to treat psoriasis(Gordon, K B et al., N Engl J Med 373:136-144 (2015). The efficacy ofguselkumab continued to increase beyond week 16 (primary end-pointassessment) and was maintained through week 40. Moreover, the majorityof patients in the 100-mg guselkumab group had completely clearedpsoriasis, as indicated by a PGA score of 0 (in 62% of patients) and a100% improvement from baseline in PASI score (in 54% of patients) after40 weeks of continuous treatment. Regulatory approval by the FDA and EMArelied in part on the findings of three pivotal phase III clinicaltrials VOYAGE 1, (Blauvelt, A et al. J. Am. Acad. Dermatol. 76: 405-417(2017) VOYAGE (Reich, K et al. J Am. Acad. Dermatol., 76: 418-431 (2017)and NAVIGATE (Langley, R G et al., Brit. J. Dermatol. 178:114-123(2017).

VOYAGE 1 (NCT02207231) was a phase III, randomized, double-blind,placebo- and active comparator-controlled trial conducted at 101 globalsites (December 2014-April 2016). The study comprised anactive-comparator period when guselkumab was compared with adalimumab(week 0-48) and a placebo-controlled period (weeks 0-16), after whichpatients taking placebo crossed over to receive guselkumab through week48. Guselkumab was superior to placebo and/or adalimumab for thecoprimary end points and all major secondary end points (all P<0.001).Compared with placebo, significantly higher proportions of patientstaking guselkumab achieved IGA 0/1 (6.9% vs 85.1%) and PASI 90 (2.9% vs73.3%) at week 16. Likewise, PASI 100 responses in the guselkumab groupwere significantly better than those in the adalimumab group at weeks 24and 48 (P<0.001). After initiating guselkumab at week 16, patients inthe placebo cross-over group achieved responses similar to thoseobserved in the guselkumab group. VOYAGE 1 confirms the role of IL-23 inthe pathogenesis of psoriasis. When compared with TNF-α blockade,selective targeting of the IL-23 pathway provides morepsoriasis-specific cytokine inhibition with a higher degree of efficacywhile maintaining a favorable safety profile (Blauvelt, A, et al., JInvestig Dermatol., 135: 1946-1953 (2015).

VOYAGE 1 was an extended-label trial that followed patients for fouryears after the initial trial. The patients were initially randomized toreceive either Tremfya or placebo, but at 16 weeks everyone receivedTremfya. The VOYAGE 1 study found that 82% of patients receiving Tremfyain a combined group of individuals that initially received Tremfya orplacebo then crossed over to Tremfya at week 16 showed at least a 90%improvement in the Psoriasis Area Severity Index (PASI 90) and anInvestigator's Global Assessment (IGA) score of cleared (0) or minimaldisease (1) at week 204, which is four years.

Psoriatic arthritis (PsA) is a chronic inflammatory musculoskeletaldisease that occurs in up to 40% of patients with psoriasis.Consequently, PsA can be considered as a disease within a disease,sharing many common pathogenic pathways with psoriasis. Psoriasisusually precedes PsA in 70% of patients, with inflammatory skin andjoint disease occurring simultaneously in 15% of patients and theinflammatory arthritis occurring before the dermatosis in the remainingpatients Eventually, almost all patients with PsA will developpsoriasis, however the clinical presentation and course of PsA is quiteheterogeneous and five distinct patterns of PsA based on thedistribution of afflicted joints have been described (Dobbin-Sears, I etal. Ther Adv Chronic Dis, 9(10) 191-198 (2018)).

PsA is a heterogeneous condition with articular and extra-articularmanifestations, including a combination of peripheral arthritis, axialdisease, enthesitis, dactylitis and skin and nail disease (Quireo, R andCoto-Sequra, P, Expert Opinion On Biological Therapy, 18:9, 931-935(2018)). Genetic, immunologic and environmental factors activating boththe innate and acquired immune response appear to have an important rolein the pathogenesis of PsA. With disease evolution, patients may exhibitmultiple patterns and are not limited to one subset of arthritis.Approximately two-thirds of PsA patients will experience progressivejoint damage that is often associated with functional loss anddisability.

The pro-inflammatory IL-23/IL-23 receptor signaling axis is implicatedin both PsO and PsA. In particular, the Th-17 axis (inhibited by IL-23)is considered to play an important role in the immunopathogenesis ofboth psoriasis and PsA. The IL-23/IL-23-R interaction inducesIL-23-dependent differentiation and activation of Th-17 cells, and theproduction and secretion of IL-17 and IL-22 culminating in synovium andskin inflammation as well as bone remodeling. Of particular relevance toPsA pathology, IL-17 promotes bone erosion through the upregulation ofRANKL. Integrated data analysis results indicated thatustekinumab-treated patients (regardless of dose) significantlyinhibited radiographic progression of joint damage in patients withactive PsA (Kavanaugh, A et al. Ann. Rheum. Dis. 73(6):1000-1006 (2014).This supports the roles of IL-23 and the downstream Th17 pathway in theradiographic damage that occurs in most PsA patients.

Crohn's disease (CD) and ulcerative colitis (UC), the major inflammatorybowel diseases (IBD) in humans, are both chronically relapsing diseasescharacterized by a chronic tissue inflammation that alters the integrityand function of the gut. Increased levels of interleukin (IL)-23 and Thelper (Th) 17 cell cytokines have been found in intestinal mucosa,plasma, and serum of patients with inflammatory bowel disease (IBD)(e.g., Crohn's disease (CD) and ulcerative colitis (UC)).

Variants in several genes encoding for elements of the IL-23 and IL-17cellular pathways are associated with IBD risk. In particular, aloss-of-function variant of the IL-23 receptor gene that encodes anamino acid change from arginine to glutamine at position 381, has beenobserved to reduce the risk for IBD, attributed to decreased STAT3signaling and diminished Th17 cell responses upon exposure to IL-23(Barrett, J C et al. Nat. Genet. 40:955-962 (2008), Duerr, R H et al.Science 314:1461-1463 (2006), Allocca, M et al. Best Practice & Res.Clin. Gastro. 32-33:95-102 (2018).

Crohn's disease (CD) is a chronic immune-mediated condition that ischaracterized by a relapsing nature and involvement of thegastrointestinal system. CD is characterized by a dysregulation of bothinnate and adaptive immune responses. Although the pathophysiologicmechanisms have not been completely understood, the disease is likely aresult of the interaction between commensal flora in the gut and hostmicrobial defenses in a genetically predisposed individual, resulting ina transmural inflammatory response in Crohn's disease (Deepak, P andLoftus, E, Drug Design, Development and Therapy (10) 3685-3698) (2016).Over the long term, the persistent transmural inflammatory responseoften leads to the development of strictures and/or fistulas thatrequire hospitalization and/or surgery. After the discovery of theIL-23/IL-17 pathways the treatment paradigm for CD shifted away fromnonspecific immunosuppressive therapies (i.e., methotrexate) towardimmunotherapy targeting the IL-2 and/or/IL-17 pathways.

UC is a chronic, relapsing-remitting, inflammatory bowel disease, thatcauses continuous mucosal inflammation of the large intestine whichdevelops tiny open sores or ulcers that produce pus and mucous. It isestimated that close to 1 million patients with ulcerative colitis livein the United States and that UC affects 2.6 million people in Europe.The disease is more common among Caucasian people, although it canaffect people of any racial or ethnic group and men are more likely thanwomen to be diagnosed. The etiology of UC is poorly understood, but isbelieved to be partially attributed to an aberrant immune response tomicrobiota (microbial flora and pathogens) in subjects with a geneticpredisposition leading to chronic inflammation in the colon. Ulcerativecolitis is known to exhibit a Th2-type cytokine profile.

IL-23-specific p19 antagonists under clinical investigation for IBDinclude brazikumab (MEDI2070), risankizumab (BI 655066), mirikizumab(LY3074828), and guselkumab (Tremfya, Janssen). To date the anti-p19(anti-IL-23) antibodies, brazikumab and risankizumab have been reportedto be effective in moderate to severe CD in phase II trials.

In a phase II trial, mirikizumab, was recently shown be effective formoderate to severe UC. Across all doses studied, between 11.5 percent to22.6 percent of patients treated with mirikizumab achieved clinicalremission, compared to 4.8 percent of those treated with placebo.Additionally, greater proportions of patients treated with mirikizumabachieved endoscopic remission and symptomatic remission compared toplacebo at 12 weeks. Currently there are no p-19 selective antibodiesapproved for the treatment of IBDs. Phase 2 and 3 clinical trials ofanti-p19 agents (risankizumab, brazikumab, guselkumab) are ongoing andwill provide further information not only on their efficacy and safetyper se, but also on head-to-head efficacy compared to existing biologicsand on the evolving therapeutic concept of combination treatment withmultiple biologics.

Ankylosing spondylitis (AS), like psoriatic arthritis, is anotherspondyloarthropathy genetically associated with the IL-23 pathway; it isa painful condition involving spinal inflammation that can lead toirreversible spinal fusion. AS is generally unresponsive to conventionaldisease-modifying antirheumatic drugs (DMARDs), and systemic therapy forAS consists of non-steroidal anti-inflammatory drugs (NSAIDs) and tumornecrosis factor inhibitors.

Several lines of evidence have identified IL-23 as a promisingtherapeutic target in AS (Paine A, et al. Curr. Opin. Rheumatol.28:359-67 (2016). At the genetic level, case-control genome-wideassociation studies have demonstrated that IL-23 receptor (IL-23R)polymorphisms are associated with an increased risk of developing AS(Reveille J D, et al Genet 42:123-7 (2010). In addition, a protectiveeffect of the IL-23R R381Q polymorphism is observed in AS (Sarin R, etal. Proc Natl Acad Sci USA; 108:9560-58 (2011). Increased numbers ofIL-23-producing cells have been found in facet joints of patients withAS (Appel H, et al. Arthritis Rheum; 65:1522-9 (2013), while the numberof IL-23-responsive T helper (Th) 22 (Th22), Th17 and gamma/deltaT-cells are elevated in blood from patients with AS (Zhang L, et al.PLoS One (7):e31000 (2012).

The recent approval of the IL-17A inhibitor, secukinumab, for thetreatment of AS (Baeten D. et al., N Engl. J. Med. 373:2534-48 (2015),supported the clinical hypothesis that direct and specific inhibition ofIL-23 would be of therapeutic benefit to patients with AS. However, arecent publication reporting the results of randomized, double-blind,placebo-controlled, proof-of-concept, dose-finding phase 2 studyevaluating the efficacy of risankizumab in patients with active AS(NCT02047110), concluded that treatment with risankizumab did not meetthe study primary endpoint and showed no evidence of clinicallymeaningful improvements compared with placebo in patients with active AS(Baeten D, et al., Annals of the Rheumatic Diseases 77: 1295-1302(2018).

IL-23p19 Antagonists

The IL-23 receptor complex is comprised of IL-12Rβ1 partnered with thesignaling chain IL-23R (p19 subunit binding). IL-23 mediates cellularactivity through sequential binding to 2 receptor chains expressed asthe IL-12Rβ1/IL-23R receptor complex on the surface of T-cells andnatural killer (NK) cells.

Murine, humanized and phage display antibodies selected for inhibitionof recombinant IL-23 have been described; see for example U.S. Pat. No.7,491,391, WIPO Publications WO 1999/05280, WO 2007/0244846, WO2007/027714, WO 2007/076524, WO 2007/147019, WO 2008/103473, WO2008/103432, WO 2009/043933 and WO 2009/082624.

Monoclonal antibodies, or antigen binding domains thereof, that bindwith high affinity to the p19 subunit of the IL-23 cytokine, canneutralize its activity and consequently block its downstream effects.To date, three (3) anti-p19 antibodies, guselkumab (TREMFYA®),tildrakizumab, (ILUMYA®), and risankizumab (SKYRIZI®) have been approvedby the FDA for the treatment of IMIDs. Two other IL-23p19 subunitspecific antibodies are currently in late-stage clinical developmentMEDI2070 (brazikumab, Astrazeneca/Medimmue) and Ly3074828 (mirikizumab,Eli Lilly). Mirikizumab is a humanized IgG4 monoclonal antibody. Byblocking IL-23, anti-p19-specific antibodies inhibit the release ofpro-inflammatory cytokines and chemokines thereby dampening theinflammatory response.

Since this group of IL-23 antagonists target the p19 subunit of IL-23and not the p40 subunit, they do not affect IL-12 activity. This featuredistinguishes the IL-23 receptor antagonists from ustekinumab (STELARA)which targets the common p40 subunit shared by IL-12 and IL-23. Despitethe efficacy and favorable safety profile of ustekinumab, drugdevelopment for IMIDs has shifted its focus towards the development ofagents that selectively antagonize the IL-23/IL-17 pathways.

Guselkumab (CNTO1959) is a fully human monoclonal IgG1, λ antibody thatbinds with high affinity to the p19 subunit of human IL-23. Guselkumabis the first FDA-approved anti-p19-specific antibody/IL-23 antagonist.It was approved on Jul. 13, 2017, for the treatment of adults withmoderate-to-severe plaque psoriasis, after an expedited regulatoryreview. It has also been approved in Canada, the European Union, Japanand several other countries worldwide. Guselkumab is marketed by Janssenas TREMFYA (U.S. Pat. Nos. 7,935,344 and 7,993,645).

Guselkumab inhibits the bioactivity of human IL-23 by preventing IL-23from binding to the IL-23 receptor protein expressed on the surface ofimmune cells. More specifically, guselkumab binds to the human IL-23cytokine via the p19 subunit and prevents an IL-23-IL-23R complexformation and subsequent intracellular signaling of the partner receptorchains.

The TREMFYA® development program currently includes Phase III trialsevaluating the efficacy of TREMFYA® for treating active psoriaticarthritis, a Phase IIb/III study in Crohn's disease, a Phase IIb/IIItrial in ulcerative colitis, and another clinical study evaluatingguselkumab for Hidradenitis suppurativa.

Janssen recently announced plans to further expanded the clinicaldevelopment of guselkumab into familial adenomatous polyposis (FAP), adisease of the gastrointestinal tract. Janssen has initiated a phase Ibproof-of-concept clinical trial (NCT03649971) that will evaluate theefficacy and safety of guselkumab vs. placebo in approximately 72patients. FAP Syndrome is the most common adenomatous polyposissyndrome. It is an autosomal dominantly inherited disorder characterizedby the early onset of hundreds to thousands of adenomatous polypsthroughout the colon. FAP has a birth incidence of about 1 out of 8,300worldwide, manifests equally in both sexes, and, if left untreated,patients with this syndrome will most likely develop colorectal cancer.In addition, an increased risk exists for the development of othermalignancies. Removing the colon is currently the only way to preventcolorectal cancer from developing in these patients.

Tildrakizumab (MK322) is a is a humanized monoclonal IgG1, κ antibodymarketed by Merck & Co./Sun Pharmaceutical as ILUMYA (U.S. Pat. No.8,404,813). Tildrakizuab selectively binds to the p19 subunit, therebyinhibiting the interaction of IL-23 with its receptor, and thus inhibitsthe release of IL-23 mediated proinflammatory cytokines. It received itsfirst global approval in March 2018 from the FDA for use in adultpatients with moderate to severe plaque psoriasis.

Risankizumab (BI 655066) is a humanized monoclonal IgG1, κ marketed byAbbVie/Boehringer Ingelheim as SKYRIZI (U.S. Pat. No. 8,778,346).Risankizumab was developed as a high-affinity antibody antagonist ofIL-23.

Risankizumab selectively binds, with high affinity (dissociationconstant <10 pmol/L), to the p19 subunit of interleukin-23(IL-23p19)(Singh, S, et al., MAbs 7(4)77-791 (2015). It selectivelytargets the p19 subunit of IL-23 and potently inhibits IL-23-induced(human IL-23 produced by THP-1 cells) IL-17 production in a mousesplenocyte assay with IC50 value of approximately 2 pM ((Singh, S, etal., MAbs 7(4)77-791 (2015). The framework region of risankizumab hasbeen engineered with two mutations in the Fc region to reduce FcγRreceptor and complement binding. More specifically, the Fc portion ofrisankizumab has two replacement mutations (Leu234Ala and Leu235Ala) toreduce Fcγ receptor and complement binding. Risankizumab was approved bythe FDA for the treatment of moderate to severe plaque psoriasis inadults in April 2019.

Anti-IL-23p19 Antibodies

The anti-IL-23p19 antibodies of the disclosure bind to the p19 subunitof IL-23. Preferably, such antibodies are fully human and do not bindthe p40 subunit of IL-12.

In an embodiment, the anti-IL-23p19 antibodies or antibody fragmentsthereof comprise a VH having a set of CDRs (HCDR1, HCDR2, and HCDR3)disclosed in Table 1. For example, the anti-IL-23p19 antibodies orantibody fragments thereof may comprise a set of CDRs corresponding tothose CDRs in one or more of the anti-IL-23p19 antibodies disclosed inTable 1 (e.g., the CDRs of the Hu-2. 18006B antibody).

In another embodiment, the anti-IL-23p19 antibodies comprise a VL havinga set of CDRs (LCDR1, LCDR2, and LCDR3) as disclosed in Table 2. Forexample, the anti-IL-23p19 antibodies or antibody fragments thereof maycomprise a set of CDRs corresponding to those CDRs in one or more of theanti-IL-23p19 antibodies disclosed in Table 2 (e.g., the CDRs of theHu-2. 18006B antibody).

In an alternative embodiment, the anti-IL-23p19 antibodies or antibodyfragments thereof comprise a VH having a set of CDRs (HCDR1, HCDR2, andHCDR3) as disclosed in Table 1, and a VL having a set of CDRs (LCDR1,LCDR2, and LCDR3) as disclosed in Table 2.

TABLE 1 CDR Sequences of Human Variable Heavy Chain DomainsAnti-IL-23p19 Ab CDR1 CDR2 CDR3 Hu-2. 18006B SEQ ID NO: 9  SEQ ID NO: 10SEQ ID NO: 11 Hu-4. 18006B SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO: 17Hu-5. 18006B SEQ ID NO: 21 SEQ ID NO: 22 SEQ ID NO: 23 Hu-6. 18006B SEQID NO: 27 SEQ ID NO: 28 SEQ ID NO: 29

TABLE 2 CDR Sequences of Human Variable Light Chain DomainsAnti-IL-23p19 Ab CDR1 CDR2 CDR3 Hu-2. 18006B SEQ ID NO: 12 SEQ ID NO: 13SEQ ID NO: 14 Hu-4. 18006B SEQ ID NO: 18 SEQ ID NO: 19 SEQ ID NO: 20Hu-5. 18006B SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 26 Hu-6. 18006B SEQID NO: 30 SEQ ID NO: 31 SEQ ID NO: 32

In an embodiment, the antibody may be a monoclonal, chimeric, humanizedor human antibody (or antigen-binding portions thereof) thatspecifically bind to human IL-23p19.

In an embodiment, the anti-IL-23p19 antibodies or antibody fragmentsthereof comprise a VH having a set of complementarity-determiningregions (CDR1, CDR2, and CDR3) selected from the group consisting of:

-   -   (i) CDR1: SEQ ID NO: 9, CDR2: SEQ ID NO: 10, CDR3: SEQ ID NO:        11;    -   (ii) CDR1: SEQ ID NO: 15, CDR2: SEQ ID NO: 16, CDR3: SEQ ID NO:        17;    -   (iii) CDR1: SEQ ID NO: 21, CDR2: SEQ ID NO: 22, CDR3: SEQ ID NO:        23; and    -   (iv) CDR1: SEQ ID NO: 27, CDR2: SEQ ID NO: 28, CDR3: SEQ ID NO:        29.

In another embodiment, the anti-IL-23p19 antibodies or antibodyfragments thereof comprise a VL having a set ofcomplementarity-determining regions (CDR1, CDR2, and CDR3) selected fromthe group consisting of:

-   -   (i) CDR1: SEQ ID NO: 12, CDR2: SEQ ID NO: 13, CDR3: SEQ ID NO:        14;    -   (ii) CDR1: SEQ ID NO: 18, CDR2: SEQ ID NO: 19, CDR3: SEQ ID NO:        20;    -   (iii) CDR1: SEQ ID NO: 24, CDR2: SEQ ID NO: 25, CDR3: SEQ ID NO:        26; and    -   (iv) CDR1: SEQ ID NO: 30, CDR2: SEQ ID NO: 31, CDR3: SEQ ID NO:        32.

In another embodiment, the anti-IL-23p19 antibodies or antibodyfragments thereof comprise:

(a) a VH having a set of complementarity-determining regions (CDR1,CDR2, and CDR3) selected from the group consisting of:

-   -   (i) CDR1: SEQ ID NO: 9, CDR2: SEQ ID NO: 10, CDR3: SEQ ID NO:        11;    -   (ii) CDR1: SEQ ID NO: 15, CDR2: SEQ ID NO: 16, CDR3: SEQ ID NO:        17;    -   (iii) CDR1: SEQ ID NO: 21, CDR2: SEQ ID NO: 22, CDR3: SEQ ID NO:        23; and    -   (iv) CDR1: SEQ ID NO: 27, CDR2: SEQ ID NO: 28, CDR3: SEQ ID NO:        29; and

(b) a VL having a set of complementarity-determining regions (CDR1,CDR2, and CDR3) selected from the group consisting of:

-   -   (i) CDR1: SEQ ID NO: 12, CDR2: SEQ ID NO: 13, CDR3: SEQ ID NO:        14;    -   (ii) CDR1: SEQ ID NO: 18, CDR2: SEQ ID NO: 19, CDR3: SEQ ID NO:        20;    -   (iii) CDR1: SEQ ID NO: 24, CDR2: SEQ ID NO: 25, CDR3: SEQ ID NO:        26; and    -   (iv) CDR1: SEQ ID NO: 30, CDR2: SEQ ID NO: 31, CDR3: SEQ ID NO:        32.

In an embodiment, the antibodies comprise a combination of a VH and a VLhaving a set of complementarity-determining regions (CDR1, CDR2 andCDR3) selected from the group consisting of:

-   -   (i) VH: CDR1: SEQ ID NO: 9, CDR2: SEQ ID NO: 10, CDR3: SEQ ID        NO: 11, VL: CDR1: SEQ ID NO: 12, CDR2: SEQ ID NO: 13, CDR3: SEQ        ID NO: 14;    -   (ii) VH: CDR1: SEQ ID NO: 15, CDR2: SEQ ID NO: 16, CDR3: SEQ ID        NO: 17, VL: CDR1: SEQ ID NO: 18, CDR2: SEQ ID NO: 19, CDR3: SEQ        ID NO: 20;    -   (iii) VH: CDR1: SEQ ID NO: 21, CDR2: SEQ ID NO: 22, CDR3: SEQ ID        NO: 23, VL: CDR1: SEQ ID NO: 24, CDR2: SEQ ID NO: 25, CDR3: SEQ        ID NO: 26;    -   and    -   (iv) VH: CDR1: SEQ ID NO: 27, CDR2: SEQ ID NO: 28, CDR3: SEQ ID        NO: 29, VL: CDR1: SEQ ID NO: 30, CDR2: SEQ ID NO: 31, CDR3: SEQ        ID NO: 32.

In an embodiment, the anti-IL-23p19 antibodies or antibody fragmentsthereof comprise a variable heavy chain sequence selected from the groupconsisting of: SEQ ID NOs: 1, 3, 5, and 7; and/or a variable light chainsequence selected from the group consisting of: SEQ ID NOs: 2, 4, 6, and8.

In an embodiment, the anti-IL-23p19 antibodies or antibody fragmentsthereof comprise a pair of variable heavy chain and variable light chainsequences, selected from the following combinations: a variable heavychain sequence comprising SEQ ID NO: 1 and a variable light chainsequence comprising SEQ ID NO: 2; a variable heavy chain sequencecomprising SEQ ID NO: 3 and a variable light chain sequence comprisingSEQ ID NO: 4; a variable heavy chain sequence comprising SEQ ID NO: 5and a variable light chain sequence comprising SEQ ID NO: 6; and avariable heavy chain sequence comprising SEQ ID NO: 7 and a variablelight chain sequence comprising SEQ ID NO: 8. The skilled person willfurther understand that the variable light and variable heavy chains maybe independently selected, or mixed and matched, to prepare ananti-IL-23p19 antibody comprising a combination of variable heavy andvariable light chain that is distinct from the pairings identifiedabove.

In an embodiment, the anti-IL-23p19 antibodies or antibody fragmentsthereof comprise a pair of variable heavy chain and variable light chainsequences, selected from the following combinations: a variable heavychain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 1 and avariable light chain sequence that is 90%, 95%, or 99% identical to SEQID NO: 2; a variable heavy chain sequence that is 90%, 95%, or 99%identical to SEQ ID NO: 3 and a variable light chain sequence that is90%, 95%, or 99% identical to SEQ ID NO: 4; a variable heavy chainsequence that is 90%, 95%, or 99% identical to SEQ ID NO: 5 and avariable light chain sequence that is 90%, 95%, or 99% identical to SEQID NO: 6; and a variable heavy chain sequence that is 90%, 95%, or 99%identical to SEQ ID NO: 7 and a variable light chain sequence that is90%, 95%, or 99% identical to SEQ ID NO: 8. The skilled person willfurther understand that the variable light and variable heavy chains maybe independently selected, or mixed and matched, to prepare ananti-IL-23p19 antibody comprising a combination of variable heavy andvariable light chain that is distinct from the pairings identifiedabove.

In some embodiments, the anti-IL-23p19 antibodies (e.g., antagonistantibodies) bind with high affinity to the p19 subunit of IL-23 and donot bind to the p40 subunit of the related cytokine family member,IL-12.

In some embodiments, the antibody is a full-length antibody. In otherembodiments, the antibody is an antibody fragment including, forexample, an antibody fragment selected from the group consisting of:Fab, Fab′, F(ab)₂, Fv, domain antibodies (dAbs), and complementaritydetermining region (CDR) fragments, single-chain antibodies (scFv),chimeric antibodies, diabodies, triabodies, tetrabodies, miniantibodies,and polypeptides that contain at least a portion of an immunoglobulinthat is sufficient to confer IL-23 specific binding to the polypeptide.

In some embodiments, a variable region domain of an anti-IL-23p19antibody disclosed herein may be covalently attached at a C-terminalamino acid to at least one other antibody domain or a fragment thereof.Thus, for example, a VH domain that is present in the variable regiondomain may be linked to an immunoglobulin CH1 domain, or a fragmentthereof. Similarly, a VL domain may be linked to a CK domain or afragment thereof. In this way, for example, the antibody may be a Fabfragment wherein the antigen binding domain contains associated VH andVL domains covalently linked at their C-termini to a CH1 and CK domain,respectively. The CH1 domain may be extended with further amino acids,for example to provide a hinge region or a portion of a hinge regiondomain as found in a Fab fragment, or to provide further domains, suchas antibody CH2 and CH3 domains.

In some embodiments, a variable region domain of an anti-IL-23p19antibody may be covalently attached at a C-terminal amino acid to atleast one other antibody domain or a fragment thereof. Thus, forexample, a VH domain that is present in the variable region domain maybe linked to an immunoglobulin CH1 domain, or a fragment thereof.Similarly, a VL domain may be linked to a CK domain or a fragmentthereof. In this way, for example, the antibody may be a Fab fragmentwherein the antigen binding domain contains associated VH and VL domainscovalently linked at their C-termini to a CH1 and CK domain,respectively. The CH1 domain may be extended with further amino acids,for example to provide a hinge region or a portion of a hinge regiondomain as found in a Fab′ fragment, or to provide further domains, suchas antibody CH2 and CH3 domains.

Thus, in one embodiment, the antibody fragment comprises at least oneCDR as described herein. The antibody fragment may comprise at leasttwo, three, four, five, or six CDRs as described herein. The antibodyfragment further may comprise at least one variable region domain of anantibody described herein. The variable region domain may be of any sizeor amino acid composition and will generally comprise at least one CDRsequence responsible for binding to human IL-23p19, for example, CDR-H1,CDR-H2, CDR-H3, CDR-L1, CDR-L2, and/or CDR-L3 as described herein, andwhich is adjacent to or in frame with one or more framework sequences.

In some embodiments, the anti-IL-23p19 antibody is a monoclonalantibody. In some embodiments, the anti-IL-23p19 antibody is a humanantibody. In alternative embodiments, the anti-IL-23p19 antibody is amurine antibody. In some embodiments, the anti-IL-23p19 antibody is achimeric antibody, a bispecific antibody, or a humanized antibody.

In a further aspect, the anti-IL-23p19 antibody or antibody fragmentthereof exhibits one or more of the following properties:

-   -   (a) is specific for human IL-23p19 and has the ability to block        IL-23 binding to its receptor (IL-23R);    -   (b) inhibits, interferes with, or modulates IL-23p19 interaction        with IL-23 receptor signal transduction;    -   (c) inhibits STAT3 activation induced by IL-23;    -   (d) inhibits IL-17 production induced by human IL-23 in mouse        splenocytes;    -   (e) inhibits IL-17 production induced by human IL-23 in        activated human T cells in PBMC;    -   (f) does not inhibit IL-23 interaction with IL-12Rβ1 signal        transduction;    -   (g) does not inhibit human IL-12 induced interferon gamma        production in human activated T-cells (PBMC);    -   (h) does not inhibit cynomolgus monkey IL-12 induced interferon        gamma production in human activated T-cells (PBMC); and    -   (i) inhibits skin inflammation induced by human IL-23 in a        murine psoriasis-like model.

In an embodiment, the anti-IL-23p19 antibodies or antibody fragmentsthereof can reduce, inhibit, interfere with, and/or modulate at leastone of the biological responses related to IL-23, and as such, areuseful for ameliorating the effects of IL-23 related diseases ordisorders. Such antibodies and antibody fragments thereof can be used,for example, to reduce, inhibit, interfere with and/or modulate IL-23signaling, IL-23 activation of Th17 cells, IL-23 activation of NK cells,or inducing production of proinflammatory cytokines.

In some embodiments, the anti-IL-23p19 antibodies or antibody fragmentsthereof comprise one or more conservative amino acid substitutions. Aperson of skill in the art will recognize that a conservative amino acidsubstitution is a substitution of one amino acid with another amino acidthat has similar structural or chemical properties, such as, forexample, a similar side chain. Exemplary conservative substitutions aredescribed in the art, for example, in Watson et al., Molecular Biologyof the Gene, The Benjamin/Cummings Publication Company, 4th Ed. (1987).

“Conservative modifications” refer to amino acid modifications that donot significantly affect or alter the binding characteristics of theantibody containing the amino acid sequences. Conservative modificationsinclude amino acid substitutions, additions and deletions. Conservativesubstitutions are those in which the amino acid is replaced with anamino acid residue having a similar side chain. The families of aminoacid residues having similar side chains are well defined and includeamino acids with acidic side chains (e.g., aspartic acid, glutamicacid), basic side chains (e.g., lysine, arginine, histidine), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine), uncharged polar side chains (e.g., glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine,tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan,histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine,valine, leucine, isoleucine, serine, threonine), amide (e.g.,asparagine, glutamine), beta-branched side chains (e.g., threonine,valine, isoleucine) and sulfur-containing side chains (cysteine,methionine). Furthermore, any native residue in the polypeptide may alsobe substituted with alanine, as has been previously described foralanine scanning mutagenesis (MacLennan et al. (1998) Acta Physiol ScandSuppl 643: 55-67; Sasaki et al. (1998) Adv Biophys 35: 1-24). Amino acidsubstitutions to the antibodies of the invention may be made by knownmethods for example by PCR mutagenesis (U.S. Pat. No. 4,683,195).

In one embodiment, the anti-IL-23p19 antibody or antibody fragmentthereof comprises all six of the CDR regions of the Hu-2. 18006B, Hu-4.18006B, Hu-5. 18006B, or Hu-6. 18006B antibodies formatted as a chimericor a humanized antibody. In other embodiments, the anti-IL-23p19antibody or antibody fragment thereof comprises all six of the CDRregions of one of the disclosed fully human antibodies.

Methods of Producing Antibodies

Anti-IL-23p19 antibodies or antibody fragments thereof may be made byany method known in the art. For example, a recipient may be immunizedwith soluble recombinant human IL-23 protein, or a fragment or a peptideconjugated with a carrier protein thereof. Any suitable method ofimmunization can be used. Such methods can include adjuvants, otherimmune stimulants, repeat booster immunizations, and the use of one ormore immunization routes.

Any suitable source of human IL-23 can be used as the immunogen for thegeneration of the non-human or human anti-IL-23p19 antibodies of thecompositions and methods disclosed herein.

Different forms of the IL-23 antigen may be used to generate theantibody that is sufficient to generate a biological activity. Thus, theeliciting IL-23 antigen may be a single epitope, multiple epitopes, orthe entire protein alone or in combination with one or moreimmunogenicity enhancing agents. In some aspects, the eliciting antigenis an isolated soluble full-length protein, or a soluble proteincomprising less than the full-length sequence (e.g., immunizing with apeptide comprising particular portion or epitopes of IL-23). As usedherein, the term “portion” refers to the minimal number of amino acidsor nucleic acids, as appropriate, to constitute an immunogenic epitopeof the antigen of interest. Any genetic vectors suitable fortransformation of the cells of interest may be employed, including, butnot limited to adenoviral vectors, plasmids, and non-viral vectors, suchas cationic lipids.

It is desirable to prepare monoclonal antibodies (mAbs) from variousmammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies maybe found in, e.g., Sties et al. (eds.) BASIC AND CLINICAL IMMUNOLOGY(4^(th) ed.) Lance Medical Publication, Los Altos, Calif., andreferences cited therein; Harlow and Lane (1988) ANTIBODIES: ALABORATORY MANUAL CSH Press; Goding (1986) MONOCLONAL ANTIBODIES:PRINCIPLES AND PRACTICE (2^(nd) ed.) Academic Press, New York, N.Y.Typically, spleen cells from an animal immunized with a desired antigenare immortalized, commonly by fusion with a myeloma cell. See Kohler andMilstein (196) Eur. J. Immunol. 6:511-519. Alternative methods ofimmortalization include transformation with Epstein Barr Virus,oncogene, or retroviruses, or other methods known in the art. See. e.g.,Doyle et al. (eds. 1994 and periodic supplements) CELL AND TISSUECULTURE: LABORATORY PROCEDURES, John Wiley and Sons, New York, N.Y.Colonies arising from single immortalized cells are screened forproduction of antibodies of the desired specificity and affinity for theantigen, and yield of the monoclonal antibodies produced by such cellsmay be enhanced by various techniques, including injection into theperitoneal cavity of a vertebrate host. Alternatively, one may isolateDNA sequences which encode a monoclonal antibody or an antigen bindingfragment thereof by screening a DNA library from human B cellsaccording, e.g., to the general protocol outlined by Huse et al., (1989)Science 246: 1275-1281. Thus, antibodies may be obtained by a variety oftechniques familiar to researchers skilled in the art.

Other suitable techniques involve selection of libraries of antibodiesin phage, yeast, virus or similar vector. See e.g., Huse et al., supra;and Ward et al., (1989) Nature 341:544-546. The polypeptides andantibodies disclosed herein may be used with or without modification,including chimeric or humanized antibodies. Frequently, the polypeptidesand antibodies will be labeled by joining, either covalently ornon-covalently, a substance which provides for a detectable signal. Awide variety of labels and conjugation techniques are known and arereported extensively in both the scientific and patent literatures.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Patents teaching the use of such labels includeU.S. Pat. Nos. 3,817,837; 3,850,752; 3,996,345; 4,277,437; 4,275,149;and 4,366,241. Also, recombinant immunoglobulins may be produced, seeCabilly U.S. Pat. No. 4,816,567; and Queen et al. (1989)Proc. Nat'lAcad. Sci. USA 86: 10029-10023; or made in transgenic mice, see NilsLonberg et al., (1994), Nature 368:856-859; and Mendez et al. (1997)Nature Genetics 15: 146-156; TRANSGENIC ANIMALS AND METHODS OF USE (WO2012/62118), Medarex, Trianni, Abgenix, Ablexis, OminiAb, Harbour andother technologies.

In some embodiments, the ability of the produced antibody to bind toIL-23p19 can be assessed using standard binding assays, such as surfaceplasmon resonance (SPR), Octet (BLI), ELISA, Western Blot,immunofluorescence, flow cytometric analysis, chemotaxis assays, andcell migration assays. In some aspects, the produced antibody may alsobe assessed for its ability to inhibit IL-23 from blocking IL-23receptor β1 signal transduction, and inhibit IL-23p19 and/orIL-23p19-mediated inflammatory microenvironment successive effectsincluding inhibiting IL-23 induced Stat3 phosphorylation, IL-17production and/or IFN-7 production.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing a typical purification technique. The suitability of protein A asan affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (see, e.g., Lindmark et al., 1983 J. Immunol. Meth. 62:1-13).Protein G is recommended for all mouse isotypes and for human γ3 (see,e.g., Guss et al., 1986 EMBO J. 5:1567-1575). A matrix to which anaffinity ligand is attached is most often agarose, but other matricesare available. Mechanically stable matrices such as controlled poreglass or poly (styrenedivinyl) benzene allow for faster flow rates andshorter processing times than can be achieved with agarose. Where theantibody comprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, reverse phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, typically performed at low salt concentrations(e.g., from about 0-0.25 M salt).

Also included are nucleic acids that hybridize under low, moderate, andhigh stringency conditions, as defined herein, to all or a portion(e.g., the portion encoding the variable region) of the nucleotidesequence represented by isolated polynucleotide sequence(s) that encodean antibody or antibody fragment of the present disclosure. Thehybridizing portion of the hybridizing nucleic acid is typically atleast 15 (e.g., 20, 25, 30 or 50) nucleotides in length. The hybridizingportion of the hybridizing nucleic acid is at least 80%, e.g., at least90%, at least 95%, or at least 98%, identical to the sequence of aportion or all of a nucleic acid encoding an anti-IL-23p19 polypeptide(e.g., a heavy chain or light chain variable region), or its complement.Hybridizing nucleic acids of the type described herein can be used, forexample, as a cloning probe, a primer, e.g., a PCR primer, or adiagnostic probe.

Polynucleotides, Vectors, and Cells

Other embodiments encompass isolated polynucleotides that comprise asequence encoding an anti-IL-23p19 antibody or antibody fragmentthereof, vectors, and cells comprising the polynucleotides, andrecombinant techniques for production of the antibody. The isolatedpolynucleotides can encode any desired form of the anti-IL-23p19antibody including, for example, full length monoclonal antibodies, Fab,Fab′, F(ab′)2, and Fv fragments, diabodies, linear antibodies,single-chain antibody molecules, miniantibodies, and multispecificantibodies formed from antibody fragments.

Some embodiments include isolated polynucleotides comprising sequencesthat encode the light chain variable region of an antibody or antibodyfragment having the amino acid sequence of any of SEQ ID NOs: 2, 4, 6,and 8. Some embodiments include isolated polynucleotides comprisingsequences that encode the heavy chain variable region of an antibody orantibody fragment having the amino acid sequence of SEQ ID NOs: 1, 3, 5,and 7.

In an embodiment, the isolated polynucleotide sequence(s) encodes anantibody or antibody fragment having a light chain and a heavy chainvariable region comprising the amino acid sequences of:

-   -   (a) a variable heavy chain sequence comprising SEQ ID NO: 1 and        a variable light chain sequence comprising SEQ ID NO: 2;    -   (b) a variable heavy chain sequence comprising SEQ ID NO: 3 and        a variable light chain sequence comprising SEQ ID NO: 4;    -   (c) a variable heavy chain sequence comprising SEQ ID NO: 5 and        a variable light chain sequence comprising SEQ ID NO: 6; or    -   (d) a variable heavy chain sequence comprising SEQ ID NO: 7 and        a variable light chain sequence comprising SEQ ID NO: 8.

In another embodiment, the isolated polynucleotide sequence(s) encodesan antibody or antibody fragment having a light chain and a heavy chainvariable region comprising the amino acid sequences of:

-   -   (a) a variable heavy chain sequence that is 90%, 95%, or 99%        identical to SEQ ID NO: 1 and a variable light chain sequence        that is 90%, 95%, or 99% identical to SEQ ID NO: 2;    -   (b) a variable heavy chain sequence that is 90%, 95%, or 99%        identical to SEQ ID NO: 3 and a variable light chain sequence        that is 90%, 95%, or 99% identical to SEQ ID NO: 4;    -   (c) a variable heavy chain sequence that is 90%, 95%, or 99%        identical to SEQ ID NO: 5 and a variable light chain sequence        that is 90%, 95%, or 99% identical to SEQ ID NO: 6; or    -   (d) a variable heavy chain sequence that is 90%, 95%, or 99%        identical to SEQ ID NO: 7 and a variable light chain sequence        that is 90%, 95%, or 99% identical to SEQ ID NO: 8.

The polynucleotide(s) that comprise a sequence encoding an anti-IL-23p19antibody or antibody fragment thereof can be fused to one or moreregulatory or control sequence, as known in the art, and can becontained in suitable expression vectors or cells as known in the art.Each of the polynucleotide molecules encoding the heavy or light chainvariable domains can be independently fused to a polynucleotide sequenceencoding a constant domain, such as a human constant domain, enablingthe production of intact antibodies. Alternatively, polynucleotides, orportions thereof, can be fused together, providing a template forproduction of a single chain antibody.

For recombinant production, a polynucleotide encoding the antibody isinserted into a replicable vector for cloning (amplification of the DNA)or for expression. Many suitable vectors for expressing the recombinantantibody are available. The vector components generally include, but arenot limited to, one or more of the following: a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence.

The anti-IL-23p19 antibodies or antibody fragments thereof can also beproduced as fusion polypeptides, in which the antibody or fragment isfused with a heterologous polypeptide, such as a signal sequence orother polypeptide having a specific cleavage site at the amino terminusof the mature protein or polypeptide. The heterologous signal sequenceselected is typically one that is recognized and processed (i.e.,cleaved by a signal peptidase) by the cell. For prokaryotic cells thatdo not recognize and process the anti-IL-23p19 antibody signal sequence,the signal sequence can be substituted by a prokaryotic signal sequence.The signal sequence can be, for example, alkaline phosphatase,penicillinase, lipoprotein, heat-stable enterotoxin II leaders, and thelike. For yeast secretion, the native signal sequence can besubstituted, for example, with a leader sequence obtained from yeastinvertase alpha-factor (including Saccharomyces and Kluyveromycesα-factor leaders), acid phosphatase, C. albicans glucoamylase, or thesignal described in WO 90/13646. In mammalian cells, mammalian signalsequences as well as viral secretory leaders, for example, the herpessimplex gD signal, can be used. The DNA for such precursor region isligated in reading frame to DNA encoding the anti-IL-23p19 antibody.

Expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2-υ. plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV, andBPV) are useful for cloning vectors in mammalian cells. Generally, theorigin of replication component is not needed for mammalian expressionvectors (the SV40 origin may typically be used only because it containsthe early promoter).

Expression and cloning vectors may contain a gene that encodes aselectable marker to facilitate identification of expression. Typicalselectable marker genes encode proteins that confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, or alternatively, are complement auxotrophicdeficiencies, or in other alternatives supply specific nutrients thatare not present in complex media, e.g., the gene encoding D-alanineracemase for Bacilli.

Non-Therapeutic Uses

The anti-IL-23p19 antibodies or antibody fragments described herein areuseful as affinity purification agents. In this process, the antibodiesare immobilized on a solid phase such a Protein A resin, using methodswell known in the art. The immobilized antibody is contacted with asample containing the IL-23p19 protein (or fragment thereof) to bepurified, and thereafter the support is washed with a suitable solventthat will remove substantially all the material in the sample except theIL-23p19 protein, which is bound to the immobilized antibody. Finally,the support is washed with another suitable solvent that will releasethe IL-23p19 protein from the antibody.

Anti-IL-23p19 antibodies or antibody fragments are also useful indiagnostic assays to detect and/or quantify IL-23p19 protein, forexample, detecting IL-23p19 expression in specific cells, tissues, orserum. The anti-IL-23p19 antibodies can be used diagnostically to, forexample, monitor the development or progression of a disease as part ofa clinical testing procedure to, e.g., determine the efficacy of a giventreatment and/or prevention regimen. Detection can be facilitated bycoupling the anti-IL-23p19 antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. See,for example, U.S. Pat. No. 4,741,900 for metal ions which can beconjugated to antibodies for use as diagnostics according to the presentdisclosure.

The anti-IL-23p19 antibodies or antibody fragments can be used inmethods for diagnosing an IL-23p19-associated disorder (e.g., a disordercharacterized by abnormal expression of IL-23p19) or to determine if asubject has an increased risk of developing an IL-23p19-associateddisorder. Such methods include contacting a biological sample from asubject with an anti-IL-23p19 antibody or antibody fragment thereof anddetecting binding of the antibody to IL-23p19. By “biological sample” isintended any biological sample obtained from an individual, cell line,tissue culture, or other source of cells potentially expressingIL-23p19. Methods for obtaining tissue biopsies and body fluids frommammals are well known in the art.

In some embodiments, the method can further comprise comparing the levelof IL-23p19 in a patient sample to a control sample (e.g., a subjectthat does not have an IL-23p19-associated disorder) to determine if thepatient has an IL-23p19-associated disorder or is at risk of developingan IL-23p19-associated disorder.

It will be advantageous in some embodiments, for example, for diagnosticpurposes to label the antibody with a detectable moiety. Numerousdetectable labels are available, including radioisotopes, fluorescentlabels, enzyme substrate labels and the like. The label may beindirectly conjugated with the antibody using various known techniques.For example, the antibody can be conjugated with biotin and any of thethree broad categories of labels mentioned above can be conjugated withavidin, or vice versa. Biotin binds selectively to avidin and thus, thelabel can be conjugated with the antibody in this indirect manner.Alternatively, to achieve indirect conjugation of the label with theantibody, the antibody can be conjugated with a small hapten (such asdigoxin) and one of the different types of labels mentioned above isconjugated with an anti-hapten antibody (e.g., anti-digoxin antibody).Thus, indirect conjugation of the label with the antibody can beachieved.

Exemplary radioisotopes labels include ³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. Theantibody can be labeled with the radioisotope, using the techniquesdescribed in, for example, Current Protocols in Immunology, Volumes 1and 2, 1991, Coligen et al., Ed. Wiley-Interscience, New York, N.Y.,Pubs. Radioactivity can be measured, for example, by scintillationcounting.

Exemplary fluorescent labels include labels derived from rare earthchelates (europium chelates) or fluorescein and its derivatives,rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin, andTexas Red are available. The fluorescent labels can be conjugated to theantibody via known techniques, such as those disclosed in CurrentProtocols in Immunology, for example. Fluorescence can be quantifiedusing a fluorimeter.

There are various well-characterized enzyme-substrate labels known inthe art (see, e.g., U.S. Pat. No. 4,275,149). The enzyme generallycatalyzes a chemical alteration of the chromogenic substrate that can bemeasured using various techniques. For example, alteration may be acolor change in a substrate that can be measured spectrophotometrically.Alternatively, the enzyme may alter the fluorescence orchemiluminescence of the substrate. Techniques for quantifying a changein fluorescence are described above. The chemiluminescent substratebecomes electronically excited by a chemical reaction and may then emitlight that can be measured, using a chemiluminometer, for example, ordonates energy to a fluorescent acceptor.

Examples of enzymatic labels include luciferases such as fireflyluciferase and bacterial luciferase (U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (such asglucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocydic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described, for example, inO'Sullivan et al., 1981, Methods for the Preparation of Enzyme-AntibodyConjugates for use in Enzyme Immunoassay, in Methods in Enzym. (J.Langone & H. Van Vunakis, eds.), Academic press, N.Y., 73: 147-166.

Examples of enzyme-substrate combinations include, for example:Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate,wherein the hydrogen peroxidase oxidizes a dye precursor such asorthophenylene diamine (OPD) or 3,3,5,5-tetramethyl benzidinehydrochloride (TMB); alkaline phosphatase (AP) with para-Nitrophenylphosphate as chromogenic substrate; and β-D-galactosidase (β-D-Gal) witha chromogenic substrate such as p-nitrophenyl-β-D-galactosidase orfluorogenic substrate 4-methylumbelliferyl-β-D-galactosidase.

In another embodiment, the anti-IL-23p19 antibody or antibody fragmentthereof is used unlabeled and detected with a labeled antibody thatbinds the anti-IL-23p19 antibody or antibody fragment thereof.

The antibodies and antibody fragments thereof described herein may beemployed in any known assay method, such as competitive binding assays,direct and indirect sandwich assays, and immunoprecipitation assays.See, e.g., Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc. 1987).

The anti-IL-23p19 antibody or antibody fragment thereof can be used toinhibit the binding of ligand to the IL-23 receptor. Such methodscomprise administering an anti-IL-23p19 antibody to a cell (e.g., amammalian cell) or cellular environment, whereby signaling mediated bythe IL-23 receptor is inhibited. These methods can be performed in vitroor in vivo. By “cellular environment” is intended the tissue, medium, orextracellular matrix surrounding a cell.

Compositions and Methods of Treatment

The disclosure also provides compositions including, for example,pharmaceutical compositions that comprise an anti-IL-23p19 antibody orantibody fragment thereof. Such compositions have numerous therapeuticuses for the treatment, prevention, or amelioration of diseases ordisorders (e.g., diseases or disorders involving a biological activitymediated by the IL-23/IL-23 receptor signaling axis) such as animmune-mediated inflammatory disorder or an autoimmune disease.

An anti-IL-23p19 antibody or antibody fragment thereof disclosed hereinis useful in the treatment of various diseases or disorders such as animmune-mediated inflammatory disorder (IMID) or an autoimmune disease.Methods for treating an IL-23 associated disorder comprise administeringa therapeutically effective amount of an anti-IL-23p19 antibody orantibody fragment thereof to a subject in need thereof. The IMID may beselected from the group consisting of, psoriasis, psoriatic arthritis,inflammatory bowel diseases (i.e., ulcerative colitis or Crohn'sdisease) ankylosing spondylitis, systemic lupus erythematosus,hidradenitis suppurativa, atopic dermatitis, and asthma.

The present disclosure also provides methods for the treatment orprevention of an IMID comprising administering a composition orformulation that comprises an anti-IL-23p19 antibody or antibodyfragment thereof, and optionally another immune-based therapy, to asubject in need thereof.

The disclosed antibodies are also useful in methods of treatment ofcancer, either alone (e.g., as monotherapies) or in combination withother immunotherapeutic agents and/or a chemotherapy.

The antibodies can be administered either alone or in combination withother compositions that are useful for treating an immune-mediatedinflammatory disorder or an autoimmune disease. In some embodiments,compositions including, for example, pharmaceutical compositions,comprising the anti-IL-23p19 antibody can further comprise a therapeuticagent, either conjugated or unconjugated to the binding agent.

In some aspects, a composition, e.g., a pharmaceutical composition isprovided that comprises one or more antibodies disclosed herein. Thepharmaceutical compositions may be formulated with pharmaceuticallyacceptable carriers or diluents as well as any other known adjuvants andexcipients in accordance with conventional techniques such as thosedisclosed in Remington: The Science and Practice of Pharmacy, 19thEdition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995.

Typically, compositions for administration by injection are solutions insterile isotonic aqueous buffer. Where necessary, the pharmaceutical canalso include a solubilizing agent and a local anesthetic such aslignocaine to ease pain at the site of the injection. Generally, theingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. Where thepharmaceutical is to be administered by infusion, it can be dispensedwith an infusion bottle containing sterile pharmaceutical grade water orsaline. Where the pharmaceutical is administered by injection, anampoule of sterile water for injection or saline can be provided so thatthe ingredients can be mixed prior to administration.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,bispecific and multispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

A composition can be administered by a variety of methods known in theart. As will be appreciated by the skilled artisan, the route and/ormode of administration will vary depending upon the desired results. Theactive compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for the preparation of such formulations are generally known tothose skilled in the art. See, e.g., Sustained and Controlled ReleaseDrug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., NewYork, 1978.

Dosage levels of the active ingredients in the pharmaceuticalcompositions may be varied so as to obtain an amount of the activeingredient which is effective to achieve the desired therapeuticresponse for a particular subject, composition, and mode ofadministration, without being toxic to the subject. The selected dosagelevel will depend upon a variety of pharmacokinetic factors includingthe activity of the particular compositions employed, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

The pharmaceutical compositions described herein may be administered ineffective amounts. An “effective amount” refers to the amount whichachieves a desired reaction or a desired effect alone or together withfurther doses. In the case of treatment of a particular disease or of aparticular condition, the desired reaction preferably relates toinhibition of the course of the disease. This comprises slowing down theprogress of the disease and, in particular, interrupting or reversingthe progress of the disease.

In some aspects, the compositions described herein are administered topatients, e.g., in vivo, to treat or prevent a variety of disorders suchas those described herein. Preferred patients include human patientshaving disorders that can be corrected or ameliorated by administeringthe agents modulate a biological activity of the IL-23/IL-23 receptorsignaling axis.

In some aspects, conventional viral and non-viral based gene transfermethods can be used to introduce nucleic acids encoding the antibodiesor derivatives thereof, as described herein, in mammalian cells ortarget tissues. Such methods can be used to administer nucleic acidsencoding the antibodies to cells in vitro. In some embodiments, thenucleic acids encoding the antibodies or derivatives thereof areadministered for in vivo or ex vivo gene therapy uses. In otherembodiments, gene delivery techniques are used to study the activity ofthe antibodies in cell based or animal models. Non-viral vector deliverysystems include DNA plasmids, naked nucleic acid, and nucleic acidcomplexed with a delivery vehicle such as a liposome. Viral vectordelivery systems include DNA and RNA viruses, which have either episomalor integrated genomes after delivery to the cell. Such methods are wellknown in the art.

Methods of non-viral delivery of nucleic acids encoding engineeredpolypeptides of the disclosure include lipofection, microinjection,biolistics, virosomes, liposomes, immunoliposomes, polycation orlipid:nucleic acid conjugates, naked DNA, artificial virions, andagent-enhanced uptake of DNA. Lipofection methods and lipofectionreagents are well known in the art (e.g., Transfectam™ and Lipofectin™).Cationic and neutral lipids that are suitable for efficientreceptor-recognition lipofection of polynucleotides include those ofFelgner, WO 91/17424, WO 91/16024. Delivery can be to cells (ex vivoadministration) or target tissues (in vivo administration). Thepreparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art.

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding the antibodies described herein take advantage of highlyevolved processes for targeting a virus to specific cells in the bodyand trafficking the viral payload to the nucleus. Viral vectors can beadministered directly to patients (in vivo) or they can be used to treatcells in vitro and the modified cells are administered to patients (exvivo). Conventional viral based systems for the delivery of polypeptidesof the disclosure could include retroviral, lentivirus, adenoviral,adeno-associated and herpes simplex virus vectors for gene transfer.Viral vectors are currently the most efficient and versatile method ofgene transfer in target cells and tissues. Integration in the hostgenome is possible with the retrovirus, lentivirus, and adeno-associatedvirus gene transfer methods, often resulting in long term expression ofthe inserted transgene. Additionally, high transduction efficiencieshave been observed in many different cell types and target tissues. Allpatents and publications identified are expressly incorporated herein byreference for the purpose of describing and disclosing, for example, themethodologies described in such publications that might be used inconnection with the disclosure. These publications are provided solelyfor their disclosure prior to the filing date of the presentapplication. Nothing in this regard should be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention or for any other reason. All statements as tothe date or representation as to the contents of these documents isbased on the information available to the applicants and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

To the extent not already indicated, it will be understood by those ofordinary skill in the art that any one of the various embodiments hereindescribed and illustrated may be further modified to incorporatefeatures shown in any of the other embodiments disclosed herein.

The broad scope of this disclosure is best understood with reference tothe following examples, which are not intended to limit the disclosuresto the specific embodiments. The specific embodiments described hereinare offered by way of example only, and the disclosure is to be limitedby the terms of the appended claims, along with the full scope of theequivalents to which such claims are entitled.

EXAMPLES General Methods

Methods for protein purification including immunoprecipitation,chromatography, and electrophoresis, are described. Coligan et al.(2000) Current Protocols in Protein Science, Vol. 1, John Wiley andSons, Inc., New York. Chemical analysis, chemical modification,post-translational modification, production of fusion proteins, andglycosylation of proteins are described. See, e.g., Coligan et al.(2000) Current Protocols in Protein Science, Vol. 2, John Wiley andSons, Inc., New York; Ausubel et al. (2001) Current Protocols inMolecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, N.Y., pp.16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life ScienceResearch, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech (2001)BioDirectory, Piscataway, N.J., pp. 384-391. Production, purification,and fragmentation of polyclonal and monoclonal antibodies are described.Coligan et al. (2001) Current Protocols in Immunology, Vol. 1, JohnWiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Harlowand Lane, supra.

Hybridoma supernatant was purified via HiTrap protein G column (GE, cat.No. 17040401) according to the manufacturer's procedures. Briefly,protein G column was equilibrated with DPBS (Gibco, cat. No. 14190-136)for 5 CV and hybridoma supernatant was loaded via syringe/infusion pump(Legato 200, KDS) at ambient temperature and 3 minutes residence time.The column was washed with 5 CV of DPBS and elution was performed with 4CV of pH 2.8 elution buffer (Fisher Scientific, cat. No. PI21004).Elution was fractionated, and fractions were neutralized with 1MTris-HCL, pH 8.5 (Fisher Scientific, cat No. 50-843-270) and assayed byA280 (DropSense96, Trinean). Peak fractions were pooled, and bufferexchanged into DPBS. Centrifugal filters (EMD Millipore, cat. No.UFC803024) were equilibrated in DPBS at 4,000×g for 2 minutes. Purifiedsample was loaded, DPBS was added and the sample was spun at 4,000×g for5-10 minutes spins until total DPBS volume reached ≥6 DV. The final poolwas analyzed by A280.

Standard methods in molecular biology are described. Maniatis et al.,(1982) Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Sambrook and Russell (2001)Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Wu (1993) Recombinant DNA, Vol. 217, AcademicPress, San Diego, Calif. Standard methods also appear in Ausbel et al.,(2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley andSons, Inc. New York, N.Y., which describes cloning in bacterial cellsand DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol.2), glycoconjugates and protein expression (Vol. 3), and bioinformatics(Vol. 4).

The sequences for the heavy and light chain variable regions forhybridoma clones were determined as described below. Total RNA wasextracted from 1-2×10⁶ hybridoma cells using the RNeasy Plus Mini Kitfrom Qiagen (Germantown, Md., USA). cDNA was generated by performing 5′RACE reactions using the SMARTer RACE 5′/3′ Kit from Takara (MountainView, Calif., USA). PCR was performed using the Q5 High-Fidelity DNAPolymerase from NEB (Ipswich, Mass., USA) to amplify the variableregions from the heavy and light chains using the Takara UniversalPrimer Mix in combination with gene specific primers for the 3′ mouseconstant region of the appropriate immunoglobulin. The amplifiedvariable regions for the heavy and light chains were run on 2% agarosegels, the appropriate bands excised and then gel purified using the MiniElute Gel Extraction Kit from Qiagen. The purified PCR products werecloned using the Zero Blunt PCR Cloning Kit from Invitrogen (Carlsbad,Calif., USA), transformed into Stellar Competent E. Coli cells fromTakara and plated onto LB Agar+50 μg/ml kanamycin plates. Direct colonySanger sequencing was performed by GeneWiz (South Plainfield, N.J.,USA). The resulting nucleotide sequences were analyzed using IMGTV-QUEST to identify productive rearrangements and analyze translatedprotein sequences. CDR determination was based on IMGT numbering.

Methods for flow cytometry, including fluorescence activated cellsorting detection systems (FACS®), are available. See, e.g., Owens etal. (1994) Flow Cytometry Principles for Clinical Laboratory Practice,John Wiley and Sons, Hoboken, N.J.; Givan (2001) Flow Cytometry, 2nded.; Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical Flow Cytometry,John Wiley and Sons, Hoboken, N.J. Fluorescent reagents suitable formodifying nucleic acids, including nucleic acid primers and probes,polypeptides, and antibodies, for use, e.g., as diagnostic reagents, areavailable. Molecular Probes (2003) Catalogue, Molecular Probes, Inc.,Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.

The positive controls (PC1 and PC2), IL-23p19 and IL-12/IL-23 p40specific antibodies, were made by a CRO (Biointron). Control antibodiescan be prepared by any suitable expression methods. For example, bycloning the antibody heavy and light chain variable regions into the293F or ExpiCHO™ expression system (ThermoFisher Scientific, Waltham,Mass.). These antibodies were used as controls to establish the bindingand functional assays described in the examples and tested alongside thedisclosed newly generated anti-IL-23p19-specific antibodies. “PC1”refers to a reference antibody, synthesized based on the VH and VLsequences reported in U.S. Pat. No. 7,935,344 (VH SEQ ID NO: 106 and VLSEQ ID NO: 116 in the '344 patent) and known to be specific for humanIL-23p19 subunit (Biointron, LOT NO: 20180926A04). The term “PC2” refersto a reference, synthesized based on the VH and VL sequences reported inU.S. Pat. No. 6,902,734 (VH SEQ ID NO: 7 and VL SEQ ID NO: 8 in the '734patent) and known to be specific for human IL-12/IL-23 p40 subunit(BIOINTRON LOT NO: 20180925A07).

Control antibodies can be made by standard methods. For example,plasmids containing the control antibodies' sequences can be transfectedusing a mammalian system (293F or ExpiCHO™) (Catalog Number: A29133,ThermoFisher Scientific, USA) according to the manufacturer's protocol.The cells are cultured at 37° C. and 8% CO₂ at day 1 and then at 32° C.and 5% CO₂ post-transfection in media provided in the kit. Antibodiesare purified by clarifying the ExpiCHO™ culture medium by centrifugationat 1,000 g for 10 minutes followed by 5,000 g for 30 minutes. Thesupernatant is then filtered using a 0.45 μm filter followed by a 0.22μm filter. Subsequently, the supernatant is subjected to affinitypurification using protein A/G resins (Life Technologies, Carlsbad,Calif.; Catalog #20424) according to the manufacturer's protocol. Priorto ELISA purification, antibody titer in the culture medium is roughlydetermined to ensure the amount of medium loaded occupied less than 80%of the resin binding capacity. After incubation, the resins are washedwith PBS and eluted with Elution Buffer (Life Technologies, Catalog#21004). The elution fractions are immediately adjusted to physiologicpH by adding Tris Buffer, pH 8.0. The purified antibodies aresubsequently subjected to buffer exchange and protein concentrationusing Amicon Ultra-15 Centrifugal Filter Unit (Life Technologies,Catalog #UFC900324) in PBS buffer. Antibody concentration is determinedby BCA Protein Assay. SDS-PAGE and Coomassie-staining is carried out totest the antibody purity. The purified protein is aliquoted and storedat −80° C. for long time storage or kept at 4° C. for immediate use.

The integrity of the antibody can be validated by SDS-PAGE followed byCoomassie staining under non-reducing vs reducing conditions; undernon-reducing condition, one dominating band around 150 kDa, whereasunder reducing conditions, two bands are observed, 50 kDa and 25 kDa.Standard techniques for characterizing ligand/receptor interactions areavailable. See, e.g., Coligan et al. (2001) Current Protocols inImmunology, Vol. 4, John Wiley, Inc., New York. Standard methods ofantibody functional characterization appropriate for thecharacterization of antibodies with particular mechanisms of action arealso well known to those of skill in the art.

Software packages and databases for determining, e.g., antigenicfragments, leader sequences, protein folding, functional domains, CDRannotation, glycosylation sites, and sequence alignments, are available.

Example 1: Generation of Anti-IL-23p19 Antibodies

Human anti-IL-23p19 specific antibodies were generated by immunizinghuman Ig transgenic mice (see, e.g., WO 2013/063391, TRIANNI® mice).

Immunization: TRIANNI® mice were immunized by injection with human IL-23recombinant protein or combination of human IL-23 protein andheterodimer of human p19 and mouse p40 protein intraperitoneally (IP),subcutaneously (SC), or via footpad or base of the tail. The immuneresponse was monitored by retroorbital bleeds. The plasma was screenedby ELISA (as described below) for activity of binding to human IL-23heterodimer. Mice with sufficient titers were used for fusions. Micewere boosted with the immunogen before sacrifice and removal of thespleen and draining lymph nodes.

Selection of mice producing anti-IL-23p19 antibodies: To select Triannimice producing p19-specific antibodies, sera from immunized mice wasscreened by ELISA for binding to recombinant human IL-23. Briefly, anELISA plate coated with recombinant human IL-23 was incubated withdilutions of serum from immunized mice for one hour at room temperature,the assay plate was washed, and specific antibody binding was detectedwith HRP-labeled anti-mouse IgG antibody. Plates was read using an ELISAreader (Biotek).

Generation of Hybridomas: To generate hybridomas producing humanantibodies of the disclosure, splenocytes and draining lymph node cellsharvested from immunized mice were fused to an appropriate immortalizedcell line, such as a mouse myeloma cell line. The resulting hybridomaswere screened for the production of p19-specific antibodies. Forexample, single cell suspensions of splenocytes and lymph node cellsfrom immunized mice were fused to equal number of Sp2/0 non-mouse IgGsecreting myeloma cells (ATCC, CRL 1581) by electrofusion. Cells wereplated in flat bottom 96-well tissue culture plates, followed by about 2weeks of incubation in selection medium (HAT medium), then switched tohybridoma culture medium. Approximately 10-14 days after cell plating,supernatants from individual wells were screened by ELISA as describedabove. The antibody-secreting hybridomas were transferred to 24-wellplates, screened again, and if still positive for anti-p19 activity, thehybridomas were subcloned by limiting dilution or sorting using a singlecell sorter. The stable subclones were then cultured in vitro togenerate small amounts of antibodies to be used for purification andcharacterization.

Hybridoma Screening: Hybridoma supernatants were tested for IL-23specific binding using human IL-23, human IL-12, and human p19/mouse p40by ELISA using the same assay used to monitor the immune response of theimmunized mice as described above.

Example 2: Binding of Anti-IL-23p19 Specific Antibodies

Binding of anti-IL-23p19-specific antibodies to IL-23 proteins wasanalyzed by Surface plasmon resonance (SPR) determined by BIAcore.Briefly, serial dilutions of the anti-IL-23p19 antibodies or the controlantibodies were captured on an anti-mouse or human Fc chip (s) that wereimmobilized on the CM5 chip using amine coupling kit (GE Healthcare,Catalog NO: BR-1000-50, LOT NO: 2087295).

The control antibodies used in the BIAcore binding assay included: PC1(known to be a p19 specific antibody, Biointron, LOT NO: 20180926A04);PC2 (a reference antibody with known specificity for the p40 subunit ofhuman IL-12 and IL-23, Biointron, LOT NO: 20180925A07); a human IgGisotype control (Invitrogen, Catalog NO: 02-7102, LOT NO: TJ276309); amouse IgG2a isotype control (made by Novarock Biotherapeutics) and ahuman IgG4 isotype control (Dendritics, Catalog No: DDXCH04P-100; Batch:DDXCH04-028) as negative controls.

Next, serial dilutions of IL-23 recombinant protein, human p19/mouse p40heterodimer protein, human IL-12 protein and human p40 subunit proteinin the running buffer containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA,0.005% Tween 20, pH 7.4, were injected at 50 μl/minute over immobilizedantibody for 1 minute followed by 2 minutes dissociation. Each injectionwas followed by a regeneration step with a 60-second pulse of 10 mMGlycine-HCl, pH 1.7 buffer. Fitting of experimental data was done withBIAevaluation software (GE Healthcare), fit with a Langmuir 1:1 model todetermine apparent binding.

The binding profiles of the purified antibodies are depicted in FIG. 2.FIG. 2A shows the binding profile of Hu-2 18006B (purified fromhybridoma). FIG. 2B shows the binding profile of Hu-5 18006B (purifiedfrom hybridoma). FIG. 2C shows the binding profile of Hu-6 18006B*(recombinant mIgG2a). FIG. 2D shows the binding profile of Hu-4 18006B*(*recombinant, mIgG2a). FIG. 2E shows the binding profile of Hu-418006B** (**recombinant, hIgG4). Table 3 summarizes the anti-IL-23p19antibodies and their binding specificity to human IL-23 and the humanp19/mouse p40 heterodimer protein by BIAcore. Recombinant antibodies aredesignated with astericks in the table.

TABLE 3 Binding of IL-23p19 specific antibodies by BIAcore hp19/Antibody Isotype hIL23 mp40 hIL12 hp40 Hu-2 18006B mIgG1, kappa + + − −Hu-5 18006B mIgG2b, lambda + + − − Hu-6 18006B* mIgG2a, kappa + + − −Hu-4 18006B* mIgG2a, kappa + + − − Hu-4 18006B** hIgG4, kappa + + − −PC1 (p19 hIgG1, lambda + + − − specific) PC2 (p40 hIgG1, kappa + − + +specific) hIgG hIgG − − − − mIgG2a mIgG2a − − − − *1L-23p19 recombinantantibodies; PC1 and PC2 are recombinant antibodies; the rest of IL-23p19Abs are purified from hybridomas

The results show that the anti-IL-23p19 antibodies bind to human IL-23and the human p19/mouse p40 heterodimer but not human IL-12 or human p40subunit (FIG. 2 and Table 3).

PC1 was positive on human IL-23, human p19/mouse p40 and negative onhuman IL-12 and human p40 subunit (data not showed). PC2 was positive onhuman IL-23, human IL-12, human p40 subunit and negative on humanp19/mouse p40 heterodimer. The isotype controls mIgG2a and hIgG did notbind hIL-23, hp19/mp40, hIL-12 and hp40 subunit.

Results: Anti-IL-23p19-specific antibodies were characterized by theirbinding to human IL-23 and a recombinant protein comprising aheterodimer consisting of a human p19 and a mouse p40 subunit. Theseantibodies did not bind to human IL-12 and human p40 subunit by BIAcore.

The binding specificity of the disclosed anti-IL-23p19 antibodies werealso assessed by ELISA. Briefly, biotinylated IL-23 were captured viastreptavidin coated ELISA plates. Human p19/mouse p40, human IL-12 andhuman p40 subunit were directly coated to ELISA plates. Purifiedantibodies were then added to the plates followed by detection bygoat-anti-mouse IgG-HRP (Jackson ImmunoResearch, catalog no:115-036-071, LOT NO: 147271). After addition of ABTS substrate (MossInc., catalog no: ABTS-1000, LOT NO: 03086202), ELISA plates were readusing an ELISA plate reader (Biotek). The controls depicted in FIG. 3:PC1 refers to a reference antibody (known to be a p19 specific antibody,Biointron, LOT NO:20180926A04); PC2 refers to a reference antibody(known to be a p40 specific antibody, Biointron, LOT NO: 20180925A07);PC3 refers to a reference antibody (MT155, known as a p19 specificantibody from Mabtech, catalog no: 3457-6-100, code: 3457-6-1000), thenegative controls are human IgG4 (Dendritics, catalog no: DDXCHO4P-100,LOT NO: DDXCH04-028) and the mouse IgG2a (generated by NovarockBiotherapeutics).

FIG. 3 shows the binding activities of the disclosed p19-specificantibodies. FIGS. 3A and 3B show that Hu-4 18006B** (hIgG4) and Hu-518006B (mIgG2b), Hu-6 18006B* (mIgG2a), Hu-4 18006B* (mIgG2a) and Hu-218006B (mIgG1) bind to human IL-23 in a dose-dependent manner; thepositive control PC1 binds to IL-23 in a dose dependent manner (FIG.3A). FIG. 3C shows that the these selected representative anti-IL-23p19antibodies, H-2 1800B (mIgG1), Hu-4 18006B (mIgG2c), Hu-4 18006B*(mIgG2a) and H-6 18006B* (mIgG2a) bind to human p19/mouse p40heterodimer in a dose dependent manner. FIG. 3D shows that theseanti-IL-23p19 antibodies do not bind to hL-12 while the positive controlantibody PC2 binds to hIL-12 in a dose dependent manner. FIG. 3E showsthat these anti-IL-23p19 antibodies do not bind to the human p40 subunitwhile the positive control PC2 binds to the human p40 subunit in a doseresponding manner.

Results from FIG. 3A to 3E indicated that the anti-IL-23p19-specificantibodies are characterized by binding to human IL-23 and a recombinantprotein comprising a heterodimer consisting of the human p19 combinedwith the mouse p40 subunit by ELISA. These antibodies do not bind humanIL-12 and human p40 subunit.

To ensure accurate measurements of the KD and IC50 endpoints in bindingand functional assays, the antibodies were purified from the hybridomaculture supernatants prior to testing. The binding kinetics of thedisclosed anti-IL-23p9-specific antibodies to recombinant human IL-23was determined by Surface plasmon resonance (SPR) using BIAcore 3000system docked with a CM5 chip previously immobilized via amine couplingchemistry with anti-mouse IgG antibody (GE Cat No. BR-1008-38). Flowcell 1 remained unmodified to serve as a reference cell for substractionof systematic instrument noise and drift. Fc2-1 detection was run withdouble blanking (Fc1 and blank analyte buffer). Antibody samples werediluted to 50 μg/mL in HBS-EP (GE, catalog no: BR1001-88) and injectedat a flow rate of 10 uL/minute for 1 minute. Next, hIL-23 (R&D systems,catalog no: 1290-IL/CF) diluted to 0.156-40 nM was injected at 50μL/minute for 2 minutes, followed by 10 minutes dissociation. Data wereanalyzed in BIAEvalution software (GE Healthcare) by 1:1 binding modelwith global fit to determine apparent binding kinetics.

The binding kinetic data for the disclosed anti-IL-23p19 antibodies isprovided in Table 4. The results indicate that theanti-IL-23p19-specific antibodies bind to human recombinant IL-23 with aKD ranging from 3.84E-11 to 6.62E-11 M. PC1 (known to be a p19 specificantibody, Biointron, LOT NO: 20180926A04) had KD values ranging from3.77E-10 to 1.10E-11 over multiple runs.

TABLE 4 SPR Binding Kinetics Anti-IL-23p19 mAb ka (1/Ms) kd (1/s) KD (M)Hu-2 18006B 1.57E+06 6.54E−05 4.16E−11 Hu-5 18006B 1.49E+06 9.89E−056.62E−11 Hu-6 18006B* 4.44E+06 1.71E−04 3.84E−11 Hu-4 18006B** 2.50E+061.43E−04 5.72E−11

Example 3: Blocking IL-23 Interaction with IL-23 Receptor

The ability of disclosed p19-specific antibodies to block IL-23 bindingwith its cognate high affinity IL-23 receptor was determined by ELISA.Briefly, human IL-23 receptor was coated on a 96-well plate (2 μg/ml),then serial dilutions of the purified anti-IL-23p19 antibodies premixedwith recombinant human IL-23 (50 ng/mL) were added to the plate. After a30 minute incubation, the plate was washed. Then, biotinylated anti-p40antibody (Invitrogen ref: 13-7129-85, lot: 2028761, 1/3000 dilution) wasadded to the plate. After 30 minute incubation, the plate was washedfollowed by detection by streptavidin HRP. The plate was read using aplate reader (OD 405 nM) after addition of the ABTS substrate. Thepositive control antibody PC1 used in this blocking assay is a referenceantibody (known to be a p19 specific antibody, Biointron, LOT NO:20180926A04). The negative controls were mouse IgG1 (Novus, catalog no:NBP1-97005, LOT NO: 35613), mIgG2a (made by NovaRock Biotherapeutics),and hIgG4 (Dendritics, catalog no: DDXCHO4P-100, LOT NO: DDXCH04-028).

Results: The data in FIG. 4 showed that the disclosed anti-p19antibodies (Hu-2 18006B, Hu-6 18006B*, Hu-5 18006B and Hu-4 18006B**)blocked the interaction of human IL-23 with the human IL-23 receptor ina dose-dependent manner. The positive control PC1 also blockedIL-23/IL-23 receptor interaction in a dose responding manner. Thenegative controls hIgG4 and mIgG2a were negative in the assay.

To demonstrate the specificity of the disclosed p19-specific antibodies,a blocking assay was designed to evaluate the ability of the antibodiesto block IL-23 binding with IL-12 receptor β1.

Briefly, human IL-12 receptor β1 was coated on a 96-well plate (2μg/ml), then serial dilutions of the purified anti-IL-23p19 antibodiespremixed with recombinant human IL-23 (50 ng/ml) were added to theplate. The positive control antibody used in the blocking assay was PC2(a reference antibody with known specificity for the p40 subunit of theIL-12/IL-23 p40, Biointron Lot NO: 20180925A07) The negative control wasa mouse IgG1 (Novus, catalog no: NBP1-97005, lot 35613).

After a 30 minute incubation, the plate was washed. Then, biotinylatedanti-p40 antibody (Invitrogen ref: 13-7129-85, lot: 2028761, 1/3000dilution) was added to the wells that contained the anti-IL-23p19antibodies; the biotinylated anti-p19 antibody (Mabtech, cat no: MT155,code: 3457-6-1000) was add to the wells that contained PC2 antibody.After 30 minute incubation, the plate was washed followed by detectionby streptavidin HRP. The plate was read using a plate reader (OD 405 nM)after addition of the ABTS substrate.

Results: The data in FIG. 5 showed that of the 3 selected p19 specificantibodies (Hu-6 18006 B*, Hu-4 18006B* and Hu-4 18006 B**) did notblock the IL-23/IL-12 receptor β1 binding interaction. Similarly, thePC1 antibody also did not block the IL-23/IL-12 receptor β1 interaction(data not shown). However, the p40 control antibody (PC2) blocked theinteraction of IL-23/IL-12β1 as expected.

Example 4: Inhibition of IL-17 Production in Mouse Splenocyte Assay

It is widely known that human IL-23 binds murine IL-23R and inducesmurine IL-17 production in mouse splenocytes. Human IL-23 in thepresence of IL-2 stimulates the production of IL-17 in murinesplenocytes at very low (picomolar) concentrations, which can beinhibited by coincubation with inhibitors against either p40 or p19(Aggarwal, S., et al., 2003, J Biol Chem; 278: 1910-4; Singh et al.,2015, MAbs, July-August; 7(4): 778-791).

The ability of the disclosed anti-IL-23p19-specific antibodies toinhibit human IL-23-induced IL-17 production was evaluated in a murinesplenocyte assay (MSA). The potency for the inhibition ofhuman-IL-23-induced IL-17 production was determined.

Briefly, mouse splenocytes were isolated from a C57/BL-6 mouse using aglass homogenizer and a Ficoll Pague cells isolation kit (Ge Healthcare,catalog no: 17-5442-02) following the manufacture's procedures. Thesplenocytes were activated with IL-2 (20 ng/ml for 5×10⁶ cells/ml) for 5minutes, then human IL-23 (1.5 ng/ml) was added to the splenocytes. Theactivated splenocytes were plated out to a 96-well plate, 100 ul/well.100 ul/well of the 4 purified p19 antibodies, hu-6 18006B* (mIgG2a),Hu-4 18006B* (mIgG2a), Hu-4 18006** (hIgG4) and Hu-2 18006B (mIgG1),were added to the plates.

After 72 hour incubation, the supernatants were transferred out from theplate for IL-17 quantification assay using a quantikine ELISA kit (R&D,M1700 or SM1700). Controls included in the IL-17 MSA: PC1 (a referenceantibody known to be a p19 specific antibody, Biointron LotNO:20180926A04) as a positive control; a mouse IgG1 (Novus, catalog no:NBP 1-97005) and a human IgG4 (Dendritics, Cat: DDXCHO4P-100, Lot:DDXCH04-028) as negative controls.

Results: The data presented in FIGS. 6A and 6B support the conclusionthat the disclosed antibodies selectively neutralize the binding ofIL-23 to IL-23R and, therefore, inhibit IL-17 production in adose-dependent manner.

Example 5: Inhibition of STAT3 Activation by a Reporter Cell Assay

It is known that the receptor for IL-23 comprises an IL-12Rβ1 subunitshared in common with IL-12 receptor, partnered with IL-23R. IL-23p19selectively binds to IL-23R and signaling through IL-23R induces Januskinase 2 (JAK2) which activates STAT3, leading to the upregulation ofRORγgt and subsequent increases in the production of inflammatorycytokine IL-17 (Parham et al., J. Immunol. 168:5699-5708, 2002). Inorder to determine if the disclosed anti-p19 antibodies can inhibitSTAT3 activation, the antibodies were assessed on IL-23 induced STAT3activation by a reporter cell assay. Human IL-23 induces STAT3phosphorylation upon IL-23R binding at the surface of the human lymphomaDB cells (US2013/0172272 Example 13, and Desmet, J. et al., Nat. Commun.5:5237 (2014).

DB cells were derived from the human B cell lymphoma cell line, whichexpressed endogenous IL-23 receptors and STAT3 to provide a fullyfunctional IL-23 signaling pathway. DB assay cells were generated bystable transfection of DB cells with pGL4.47[luc2p/SIE/Hybro], whichallow the quantitative detection of bioactive human IL23 using aluciferase reporter system.

This DB assay is to measure the inhibitory activity of the disclosedanti-IL-23 antibodies on human IL-23-induced STAT3 activation. Briefly,DB cells (ATCC, CRL-2289) were cultured in growth medium (RPMI+10% FBS)for 2 days. On the day of the experiment, cells were harvested andresuspended in growth medium. A serial dilutions of the testedantibodies were prepared in the growth medium in the Low Binding384-well plate (ThermoScientific 264574) followed by adding human IL-23and incubated at room temperature for 30 minutes. The mixture of thetested antibodies and human IL-23 were then added to the plate. Thesignaling assay plate was incubated in a humidified 37° C./5% CO2incubator for 16 hours. OneGlo reagent was added and the mixture wasincubated at room temperature for 2 minutes. Luminescence was read on aBioTek Neo2 (BioTek. Winooski.Vt.) and IC50 values were determined usingGraphPad® software (GraphPad Software Inc., San Diego, Calif., USA), inwhich ratio was plotted against log-transformed antibody concentrationand IC50 values were determined using non-linear regression (curve fit)of sigmoidal dose-response. Control antibodies used in the STAT3activation assay included PC1 (PC1 is a reference antibody known to bespecific for p19, Biointron, LOT NO:20180926A04) as a positive controland mIgG2a as a negative control (generated in house).

Results: As shown in Table 5, the anti-IL-23p19-specific antibodiesevaluated in the assay inhibited STAT3 activation with IC50 valuesranging from 35.2 μM to 264.6 μM with maximal 99-100% inhibition. Thepositive control (PC1) had IC50 values ranging from 24.30 μM to 117.20μM with 98-99% inhibition from multiple experiments.

TABLE 5 Inhibition of IL-23/IL-23 receptor-mediated STAT3 activationAnti-IL-23p19 mAb IC50 pM Top % of inhibition Hu-2 18006B 157.7 97% Hu-418006B 168.5 99% Hu-5 18006B 226.4 99% Hu-6 18006B 60.5 99% Hu-6 18006B*35.2 99% Hu-4 18006B* 264.6 100%  Hu-4 18006B** 157.4 100% 

Results from FIG. 7 indicated that the disclosed selected 4anti-IL-23p19 antibodies (hu-4 18006B, hu-4-18006B*, hu-6 18006B andhu-6 18006B*) inhibited STAT3 activation in a dose dependent manner. Thepositive control (PC1) also showed inhibition of the STAT3 activation ina dose response manner as expected.

Example 6: Inhibition of IL-12-Depdendent IFN-γ Production by Human PBMC

IL-12 stimulation of PBMC is known to stimulate the production of IFN-γby NK cells and T-cells. In order to determine if the disclosed anti-p19antibodies can inhibit IFN-γ production, representative disclosed humananti-p19 antibodies were analyzed in a PMBC IL-12 stimulation assay.

Briefly, human PBMC were thawed from frozen stock and resuspended inRPMI+10% FBS containing 50 ng/ml of IL-18 (R&D, 9124-IL/CF) plate in a384 well plate. A serial dilutions of the test antibodies were preparedin the growth medium in the Low Binding 384-well plate. human IL-12 (25ng/ml) or cynomolgus monkey IL-12 (25 ng/ml) was transferred to eachwell followed by incubation at room temperature for 30 minutes. Themixture (tested antibodies+IL-12) was plated to PBMC cell plate. Thecells were incubated in a humidified 37° C./5% CO2 incubator for 48hours. Production of IFN-γ was measured by AlphaLISA (PerkinElmer,AL217C) following the manufacturer's protocol.

Control antibodies used in the human PBMC assay: PC1 (a referenceantibody known to be a p19 specific antibody, Biointron, LOTNO:20180926A04), PC2 (a reference antibody with known specificity forthe p40 subunit of the IL-12 and IL-23, Biointron, LOT NO: 20180925A07),anti-IL-23 p40 subunit Mab (Hu-19 18006*, generated in-house); mIgG2a(generated by NovaRock Biotherapeutics) and human IgG4 (Dendritics, cat:DDXCHO4P-100, lot: DDXCH04-028) negative control antibodies.

Results: As showed in FIGS. 8 and 9, anti-IL-23p19 antibodies (Hu-618006B* and Hu-4 18006B* and PC1) did not inhibit human IL-12 (FIG. 8)and cynomolgus monkey IL-12 (FIG. 9) mediated IFN-γ production by humanPBMC while the positive controls, PC2 and Hu-19 18006*, did in human(FIG. 8) and cynomolgus monkey (FIG. 9) PBMC in a dose-dependent manneras expected. This data supports the conclusion that the disclosed p19antibodies are specific to p19.

Example 7: In Vivo Efficacy of Anti-p19-Specific Antibodies in anIL-23-Induced Murine Skin Inflammation Model

The role of the IL-23/L-17 pathway as a key driver of human Psoriasis(PsO) is both well characterized and clinically validated. Animal modelsof Psoriasis (PsO) are important for our understanding of thepathophysiology of human diseases. Intradermal injection of IL-23 hasbeen used to study the IL-23 pathway in rodents and can be used toassess the pharmacology of novel small molecules/biologics in thetreatment of PsO (Stephen B. Gauld et al., J. Dermatological Science, 92(2018) 45-53).

It is known that human IL-23 binds to murine IL-23 receptor and inducesmIL-17 production and inflammation in mice. Intradermal injection ofhuman IL-23 into mouse ears to induce mouse ear inflammation has beenused for psoriasis model for characterizing biological drugs for humanPsoriasis (PsO) (Aggarwal et al., J Biol Chem 2003; 278: 1910-4; Singhet al., MAbs 2015 July-August; 7(4): 77-791).

To assess the ability of the Hu-4 18006 B (mIgG2c), Hu-4 18006 B**(hIgG4), hu-5 18006B (mIgG2b) and Hu-618006 B* (mIgG2a) p19-specificantibodies to block IL-23 function in vivo, the antibodies were testedin a human IL-23-induced murine skin inflammation model. Theserepresentative antibodies were evaluated for their ability to decreasethe inflammatory response.

In this model, recombinant human IL-23 (3 μg/10 μl/mouse/day) wasinjected into the skin (i.e., intradermally) of the mouse right ear for8 consecutive days (D0-D7) to elicit a psoriasis-like inflammatory skinreaction characterized by erythema and induration with histologicalevidence of epidermal hyperplasia, parakeratosis and localizedinflammatory infiltrate.

The mice were treated twice by intraperitoneal (i.p.) injection ofIL-23p19 antibodies (hu-4 18006B, hu-4 18006B**, hu-5 18006B, or hu-618006B*) or PC1, a reference antibody known to be a p19 specificantibody (Biointron, LOT NO: 20180926A04) according to 2 protocols. Inthe first protocol, mice received PBS control (vehicle) or antibodies.The first injection was on the day before IL-23 injection and the secondinjection was on the third day after the IL-23 injection. In the secondprotocol, mice received PBS control or antibodies. The first injectionwas one hour before the IL-23 injection and the second injection was onthe third day after the IL-23 injection (FIG. 10).

Mice were measured daily for body weight, ear thickness and inflammationscore of the ears. The inflammation scores of the ears were calculatedon day 0, day 2, day 4, day 6 and day 8, based on the followingcriteria: pinna shape (relatively normal-0; minimal change-; moderate tomarked change-2; swelling and deformity severely-3). Skin color(relatively normal-0; minimal hyperplasia-1; minor hyperplasia-2; severehyperplasia-3) and white scales (relatively normal-0; minimal-1;minor-2; obvious-3). The right ear thickness of each mouse was measuredand photographed in day 0, day 2, day 4, day 6 and day 8.

On the last day of the experiment (day 8), the animals were disposedwith carbon dioxide, the blood samples were collected, and the serumswere separated (stored in −80° C. freezer). The modeling ears werecollected and cut into two pieces: one piece was fixed in 10% neutralbuffered formalin, and other piece was frozen in liquid nitrogen andstored in −80° C. freezer.

Data were given as Mean±SEM. Statistical significance were consideredwhen the P value is less than 0.05.

The data provided in Tables 6 and 7 summarize the overall scores for theinjected ears (by adding scores of pinna shape, skin color, microvesselchange and white scales). The results indicated that the mice treatedwith the representative p19 specific antibodies experienced a diminishedinflammatory response relative to the IL-23 injection model group. Theeffect started from day 4 and continued to day 8. The effect isstatistically significant. This conclusion is apparent from both thesummary score values and the ear thickness values.

TABLE 6 The overall scores of the injected ears (x ± s, n = 10) Overallscores ## Group D0 D2 D4 D6 D8 PBS 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.3± 0.95   0.2 ± 0.42   IL-23 0.0 ± 0.00 0.0 ± 0.00 1.9 ± 1.37 9.6 ±2.37   10.8 ± 1.93   PC1 (10 mg/kg) 0.0 ± 0.00 0.0 ± 0.00  0.6 ± 0.97*1.3 ± 1.34*** 2.0 ± 2.05*** Hu-4 18006B (10 mg/kg) 0.0 ± 0.00 0.0 ± 0.00 0.6 ± 1.26* 1.7 ± 1.83*** 3.2 ± 3.05*** Hu-5 18006B (10 mg/kg) 0.0 ±0.00 0.0 ± 0.00 0.8 ± 1.23 2.1 ± 1.60*** 4.1 ± 2.33*** *p < 0.05, **p <0.01, ***p < 0.001 vs. Model.

TABLE 7 The overall scores of the ears injected with IL-23 (x ± s, n =8) Overall scores ## Group D0 D2 D4 D6 D8 PBS 0 ± 0 0 ± 0  0 ± 0*** 0 ±0*** 0 ± 0*** IL-23 0 ± 0 0 ± 0 1.88 ± 0.3   5.63 ± 0.6   7.75 ± 0.53  PC1 3 mg/kg 0 ± 0 0 ± 0  0.5 ± 0.19** 1.38 ± 0.26*** 2.25 ± 0.25*** PC-16 mg/kg 0 ± 0 0 ± 0 0.38 ± 0.18**   1 ± 0.33*** 1.75 ± 0.25*** Hu-418006B** 3 mg/kg 0 ± 0 0 ± 0  0.5 ± 0.27** 1.25 ± 0.37*** 2.13 ± 0.3*** Hu-4 18006B** 6 mg/kg 0 ± 0 0 ± 0 0.25 ± 0.16** 1.13 ± 0.3***  1.88 ±0.23*** Hu-6 18006B* 3 mg/kg 0 ± 0 0 ± 0 0.38 ± 0.18** 1.13 ± 0.35***2.13 ± 0.23*** Hu-6 18006B* 6 mg/kg 0 ± 0 0 ± 0 0.25 ± 0.16**   1 ±0.27*** 1.75 ± 0.31*** **p < 0.01, ***p < 0.001 vs. Model

As shown in Tables 8 and 9, the mouse ear thickness was reduced by thetreatment of selected anti-IL-23p19 antibodies compared to the model(IL-23 treatment). The treatment effect (in vivo inhibition ofinflammatory immune response in the skin) started from day 4 andcontinued to day 8 and the effect is statistically significant (p<0.001vs. model, IL-23 treatment).

TABLE 8 The ear thickness of the injected ears in day 0 to day 8 (x ± s,n = 10) Ear thickness (mm) Group D0 D2 D4 D6 D8 PBS 0.22 ± 0.02 0.19 ±0.01 0.22 ± 0.01 0.23 ± 0.02 0.26 ± 0.02  IL-23 0.23 ± 0.02 0.25 ± 0.010.37 ± 0.06 0.56 ± 0.09 0.65 ± 0.12  PC1 (10 mg/kg) 0.23 ± 0.02 0.23 ±0.01  0.28 ± 0.08**   0.35 ± 0.04***  0.33 ± 0.03*** hu-4 18006B (10mg/kg) 0.23 ± 0.01 0.22 ± 0.02  0.31 ± 0.04*   0.35 ± 0.03***  0.43 ±0.07*** hu-5 18006B (10 mg/kg) 0.23 ± 0.01 0.23 ± 0.02 0.35 ± 0.02  0.42± 0.11** 0.50 ± 0.15* *p < 0.05, **p < 0.01, ***p < 0.001 vs. Model.

TABLE 9 The ears thickness with the injection of IL-23 (x ± s, n = 8)Ears thickness (mm) Group D0 D2 D4 D6 D8 PBS 0.19 ± 0    0.2 ± 0*** 0.24 ± 0*** 0.23 ± 0***   0.23 ± 0.01*** IL-23 0.19 ± 0   0.24 ± 0.010.39 ± 0.01 0.48 ± 0.02   0.51 ± 0.02   PC1 3 mg/kg 0.19 ± 0 0.23 ± 00.35 ± 0**  0.33 ± 0.01*** 0.34 ± 0.01*** PC1 6 mg/kg 0.19 ± 0 0.23 ± 0 0.34 ± 0*** 0.33 ± 0.01*** 0.33 ± 0.01*** Hu-4 18006B** 3 mg/kg 0.18 ±0 0.23 ± 0  0.36 ± 0.01** 0.35 ± 0.01*** 0.36 ± 0.01*** Hu-4 18006B** 6mg/kg 0.18 ± 0 0.23 ± 0  0.35 ± 0.01** 0.34 ± 0.01*** 0.35 ± 0.01***Hu-6 18006B* 3 mg/kg 0.19 ± 0 0.23 ± 0 0.36 ± 0**  0.33 ± 0.01*** 0.35 ±0***   Hu-6 18006B* 6 mg/kg 0.19 ± 0 0.23 ± 0  0.34 ± 0*** 0.33 ±0.01*** 0.34 ± 0.01*** **p < 0.01, ***p < 0.001 vs. Model

The data provided in FIG. 10 shows that the disclosed anti-p19-specificantibodies hu-4 18006B** and hu-6 18006B* caused a statisticallysignificant decrease in ear thickness compared to the untreated control(model receiving human IL-23 treatment only).

FIGS. 11A, 11B, 11C and 11D provide the data establishing the effect ofthe anti-p19-specific antibodies on the inflammatory skin reaction, asdetermined by H&E pathology staining scores, such as the thickness ofthe epidermis (FIG. 11A), thickness of the dermis (FIG. 11B),infiltration of inflammatory cells (FIG. 11C) and hyperkeratosis orinsufficiency (FIG. 11D) obtained over the course of the experiment andrepresented by a score system as described below.

Briefly, on day 8, the mouse ears were collected and observedmicroscopically. The tissues were fixed in 10% neutral bufferedformalin. After fixation, the tissues were trimmed, dehydrated,embedded, sectioned into slides and stained with hematoxylin/eosin (H&E)according to relevant SOPs. The study pathologist performedhistopathology evaluation by a light microscope. A five-step gradingsystem (relatively normal, minimal, mild, moderate to marked, severe)was used to categorize the microscopic findings.

Results: Treatment with the anti-p19 antibody hu4 18006B resulted insignificant reductions of both the swelling response and theinflammation score induced by IL-23 injection compared to the model.Hu-4 18006B shows superior inhibition effect compared to PC1 on 3 of thescores, the thickness of the epidermis (FIG. 11A), the thickness ofdermis (FIG. 11B) and infiltration of inflammatory cells (FIG. 11C).Hu-4 also showed inhibition of the hyperkeratosis (FIG. 11D).

FIG. 12 provides the representative photos of the H&E stained earsections obtained on day 8 after the treatments. The H&E stainingprocedures are as described above.

Results: the selected disclosed anti-IL-23p19 antibody treatments (Hu-418006B) significantly inhibited mouse skin inflammation compared withthe model.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent disclosure. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the disclosure are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the disclosure (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the disclosure and does not pose alimitation on the scope of the disclosure otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the disclosure.

Groupings of alternative elements or embodiments of the disclosuredisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group can be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this disclosure are described herein, includingthe best mode known to the inventors for carrying out the disclosure. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the disclosureto be practiced otherwise than specifically described herein.Accordingly, this disclosure includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedelements in all possible variations thereof is encompassed by thedisclosure unless otherwise indicated herein or otherwise clearlycontradicted by context.

Specific embodiments disclosed herein can be further limited in theclaims using “consisting of” or “consisting essentially of” language.When used in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the disclosure so claimed areinherently or expressly described and enabled herein.

It is to be understood that the embodiments of the disclosure disclosedherein are illustrative of the principles of the present disclosure.Other modifications that can be employed are within the scope of thedisclosure. Thus, by way of example, but not of limitation, alternativeconfigurations of the present disclosure can be utilized in accordancewith the teachings herein. Accordingly, the present disclosure is notlimited to that precisely as shown and described.

While the present disclosure has been described and illustrated hereinby references to various specific materials, procedures and examples, itis understood that the disclosure is not restricted to the particularcombinations of materials and procedures selected for that purpose.Numerous variations of such details can be implied as will beappreciated by those skilled in the art. It is intended that thespecification and examples be considered as exemplary only, with thetrue scope and spirit of the disclosure being indicated by the followingclaims. All references, patents, and patent applications referred to inthis application are herein incorporated by reference in their entirety.

What is claimed is:
 1. An anti-IL-23p19 antibody comprising: (a) a heavychain variable region comprising CDR1: SEQ ID NO: 9, CDR2: SEQ ID NO:10, and CDR3: SEQ ID NO: 11; and a light chain variable regioncomprising CDR1: SEQ ID NO: 12, CDR2: SEQ ID NO: 13, and CDR3: SEQ IDNO: 14; (b) a heavy chain variable region comprising CDR1: SEQ ID NO:15, CDR2: SEQ ID NO: 16, and CDR3: SEQ ID NO: 17; and a light chainvariable region comprising CDR1: SEQ ID NO: 18, CDR2: SEQ ID NO: 19, andCDR3: SEQ ID NO: 20; (c) a heavy chain variable region comprising CDR1:SEQ ID NO: 21, CDR2: SEQ ID NO: 22, and CDR3: SEQ ID NO: 23; and a lightchain variable region comprising CDR1: SEQ ID NO: 24, CDR2: SEQ ID NO:25, and CDR3: SEQ ID NO: 26; or (d) a heavy chain variable regioncomprising CDR1: SEQ ID NO: 27, CDR2: SEQ ID NO: 28, and CDR3: SEQ IDNO: 29; and a light chain variable region comprising CDR1: SEQ ID NO:30, CDR2: SEQ ID NO: 31, and CDR3: SEQ ID NO:
 32. 2. The anti-IL-23p19antibody of claim 1, wherein the antibody comprises: (a) a heavy chainvariable region sequence of SEQ ID NO: 1 and a light chain variableregion sequence of SEQ ID NO: 2; (b) a heavy chain variable regionsequence of SEQ ID NO: 3 and a light chain variable region sequence ofSEQ ID NO: 4; (c) a heavy chain variable region sequence of SEQ ID NO: 5and a light chain variable region sequence of SEQ ID NO: 6; or (d) aheavy chain variable region sequence of SEQ ID NO: 7 and a light chainvariable region sequence of SEQ ID NO:
 8. 3. The anti-IL-23p19 antibodyof claim 1, wherein the antibody is an anti-human IL-23p19 antibody. 4.The anti-IL-23p19 antibody of claim 1, wherein the antibody is afull-length antibody.
 5. The anti-IL-23p19 antibody of claim 1, whereinthe antibody is an antibody fragment.
 6. The anti-IL-23p19 antibody ofclaim 4, wherein the antibody fragment is selected from the groupconsisting of: Fab, Fab′, F(ab)2, Fd, Fv, scFv and scFv-Fc fragment, asingle-chain antibody, a minibody, and a diabody.
 7. The anti-IL-23p19antibody of claim 1, wherein the antibody is a monoclonal antibody. 8.The anti-IL-23p19 antibody of claim 1, wherein the antibody is a humanantibody.
 9. The anti-IL-23p19 antibody of claim 1, wherein the antibodyis a murine antibody.
 10. The anti-IL-23p19 antibody of claim 1, whereinthe antibody is a chimeric antibody.
 11. The anti-IL-23p19 antibody ofclaim 1, wherein the antibody is a bispecific antibody.
 12. Theanti-IL-23p19 antibody of claim 1, wherein the antibody is a humanizedantibody.
 13. The anti-IL-23p19 antibody of claim 1, wherein theantibody does not bind the p40 subunit of IL-12.
 14. A pharmaceuticalcomposition comprising the antibody of claim 1 and a pharmaceuticallyacceptable carrier.
 15. A method of treating or preventing animmune-mediated inflammatory disease, the method comprisingadministering the antibody of claim 1 to a patient in need thereof. 16.An isolated polynucleotide comprising a sequence encoding ananti-IL-23p19 antibody according to claim
 1. 17. An isolatedpolynucleotide according to claim 16, encoding a sequence as set forthin any one of SEQ ID NOs. 1, 3, 5, or
 7. 18. A vector comprising apolynucleotide according to claim
 16. 19. A cell comprising apolynucleotide according to claim 16, and/or a vector according to claim17.
 20. A method for the production of an anti-IL-23p19 antibodyaccording to claim 1, the method comprising culturing the cell of claim19.